How to overcome constraints imposed by microsporidian genome features to ensure gene prediction?

Since the advent of sequencing techniques and due to their continuous evolution, it has become easier and less expensive to obtain the complete genome sequence of any organism. Nevertheless, to elucidate all biological processes governing organism development, quality annotation is essential. In genome annotation, predicting gene structure is one of the most important and captivating challenges for computational biology. This aspect of annotation requires continual optimization, particularly for genomes as unusual as those of microsporidia. Indeed, this group of fungal-related parasites exhibits specific features (highly reduced gene sizes, sequences with high rate of evolution) linked to their evolution as intracellular parasites, requiring the implementation of specific annotation approaches to consider all these features. This review aimed to outline these characteristics and to assess the increasingly efficient approaches and tools that have enhanced the accuracy of gene prediction for microsporidia, both in terms of sensitivity and specificity. Subsequently, a final part will be dedicated to postgenomic approaches aimed at reinforcing the annotation data generated by prediction software. These approaches include the characterization of other understudied genes, such as those encoding regulatory noncoding RNAs or very small proteins, which also play crucial roles in the life cycle of these microorganisms.

Nonomuraea sediminis sp. nov., a novel actinobacterium with antimicrobial activity, isolated from sediment of Dianchi Lake.

A novel actinobacterium with antimicrobial activity, designated strain H16431T, was isolated from a sediment sample collected from Dianchi Lake, Yunnan Province, PR China. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain H16431T was most closely related to Nonomuraea rhizosphaerae CGMCC 4.7431T and Nonomuraea guangzhouensis CGMCC 4.7101T (98.1% similarity), but formed a monophyletic clade with Nonomuraea ceibae KCTC 39826T (98.0% similarity). Phylogenomic analysis based on whole-genome sequence showed that strain H16431T formed a separate clade within the genus Nonomuraea. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain H16431T and its closely related Nonomuraea species were 80.0-81.5%, 71.2-74.6%, and 23.2-25.0%, respectively, which were significantly lower than the widely accepted species-defined threshold. The DNA G + C content was 70.2% based on the whole-genome sequence. The menaquinones were identified as MK-9(H4), MK-9(H6), and MK-9(H2). The major fatty acids were iso-C16:0, 10 methyl-C17:0, and iso-C16:0 2OH. The phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, and phosphatidylinositol. These chemotaxonomic characteristics were corresponded to those of the genus Nonomuraea. On the basis of the taxonomic evidence, strain H16431T represents a novel species of the genus Nonomuraea, for which the name Nonomuraea sediminis sp. nov. is proposed. The type strain is H16431T (=JCM 34852T=CICC 25119T).

Biochemical, functional and genomic characterization of a new probiotic Ligilactobacillus salivarius F14 from the gut of tribes of Odisha.

Characterization of new potential probiotics is desirable in the field of research on probiotics for their extensive use in health and disease. Tribes could be an unusual source of probiotics due to their unique food habits and least dependence on medications and consumption of antibiotics. The aim of the present study is to isolate lactic acid bacteria from tribal fecal samples of Odisha, India, and characterize their genetic and probiotic attributes. In this context one of the catalase-negative and Gram-positive isolates, identified using 16S rRNA sequencing as Ligilactobacillus salivarius, was characterized in vitro for its acid and bile tolerance, cell adhesion and antimicrobial properties. The whole genome sequence was obtained and analyzed for strain level identification, presence of genomic determinants for probiotic-specific features, and safety. Genes responsible for its antimicrobial and immunomodulatory functions were detected. The secreted metabolites were analyzed using high resolution mass spectroscopy; the results indicated that the antimicrobial potential could be due to the presence of pyroglutamic acid, propionic acid, lactic acid, 2-hydroxyisocaproic acid, homoserine, and glutathione, and the immuno-modulating activity, contributed by the presence of short chain fatty acids such as acetate, propionate, and butyrate. So, to conclude we have successfully characterized a Ligilactobacillus salivarius species with potential antimicrobial and immunomodulatory ability. The health-promoting effects of this probiotic strain and/or its derivatives will be investigated in future.

Tabrizicola rongguiensis sp. nov., isolated from the sediment of a river in Ronggui, Foshan city, China.

A novel Gram-negative, aerobic, non-spore-forming, non-motile and rod-shaped bacterium, designated J26T, was isolated from the sediment of a river in Ronggui, Foshan city, China. Strain J26T grew optimally at 0 % (w/v) NaCl, pH 6.5-7.5, and 30 °C, and it formed milky white irregular colonies on Reasoner's 2A agar medium. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain J26T had the highest similarity to Tabrizicola aquatica RCRI19T (97.1 %) and formed a distinct clade in the genus Tabrizicola. Cellular components of J26T supported this strain as a member of the genus Tabrizicola. The predominant fatty acids were C18 : 1 ω7c, C18 : 1 ω7c-11 methyl and C16 : 0. Polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphorylethanolamine. Ubiquinone Q-10 was the major respiratory quinone, and the DNA G+C content was 64.2 mol%. However, low 16S rRNA gene sequence similarity and average nucleotide identity (73.56 % for ANIb between strain J26T with RCRI19T) demonstrated that strain J26T should be assigned to a novel species. Moreover, the differences between J26T and RCRI19T in terms of physiological and biochemical properties, such as carbon, nitrogen and sulphur metabolism, further supported that J26T represents a novel species, for which the name Tabrizicola rongguiensis sp. nov. is proposed. The type strain is J26T (=GDMCC 1.2843T=KCTC 92112T).

Mycoavidus sp. Strain B2-EB: Comparative Genomics Reveals Minimal Genomic Features Required by a Cultivable Burkholderiaceae-Related Endofungal Bacterium.

Obligate bacterial endosymbionts are critical to the existence of many eukaryotes. Such endobacteria are usually characterized by reduced genomes and metabolic dependence on the host, which may cause difficulty in isolating them in pure cultures. Family Burkholderiaceae-related endofungal bacteria affiliated with the Mycoavidus-Glomeribacter clade can be associated with the fungal subphyla Mortierellomycotina and Glomeromycotina. In this study, a cultivable endosymbiotic bacterium, Mycoavidus sp. strain B2-EB, present in the fungal host Mortierella parvispora was obtained successfully. The B2-EB genome (1.88 Mb) represents the smallest genome among the endofungal bacterium Mycoavidus cysteinexigens (2.64-2.80 Mb) of Mortierella elongata and the uncultured endosymbiont "Candidatus Glomeribacter gigasporarum" (1.37 to 2.36 Mb) of arbuscular mycorrhizal fungi. Despite a reduction in genome size, strain B2-EB displays a high genome completeness, suggesting a nondegenerative reduction in the B2-EB genome. Compared with a large proportion of transposable elements (TEs) in other known Mycoavidus genomes (7.2 to 11.5% of the total genome length), TEs accounted for only 2.4% of the B2-EB genome. This pattern, together with a high proportion of single-copy genes in the B2-EB genome, suggests that the B2-EB genome reached a state of relative evolutionary stability. These results represent the most streamlined structure among the cultivable endofungal bacteria and suggest the minimal genome features required by both an endofungal lifestyle and artificial culture. This study allows us to understand the genome evolution of Burkholderiaceae-related endosymbionts and to elucidate microbiological interactions.IMPORTANCE This study attempted the isolation of a novel endobacterium, Mycoavidus sp. B2-EB (JCM 33615), harbored in the fungal host Mortierella parvispora E1425 (JCM 39028). We report the complete genome sequence of this strain, which possesses a reduced genome size with relatively high genome completeness and a streamlined genome structure. The information indicates the minimal genomic features required by both the endofungal lifestyle and artificial cultivation, which furthers our understanding of genome reduction in fungal endosymbionts and extends the culture resources for biotechnological development on engineering synthetic microbiomes.

Brevibacterium rongguiense sp. nov., isolated from freshwater sediment.

A Gram stain-positive, non-spore-forming, non-motile and rod-shaped actinomycete, strain 5221T, was isolated from the sediment of a river collected at Ronggui in the Pearl River Delta, PR China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain formed a distinct lineage within the genus Brevibacterium and had the highest sequence similarity to Brevibacterium pityocampae Tp12T (96.7 %), followed by Brevibacterium daeguense 2C6-41T (96.5 %), Brevibacterium samyangense SST-8T (96.0 %) and Brevibacterium ravenspurgense 20T (95.9 %). The results of chemotaxonomic analyses, including detecting anteiso-C15 : 0, anteiso-C17 : 0, and C16 : 0 as the major cellular fatty acids, diphosphatidylglycerol, phosphatidylglycerol and three phosphoglycolipids as the polar lipids, MK-8(H2) as the major menaquinone, and a DNA G+C content of 72.4 mol%, supported that strain 5221T is a member of the genus Brevibacterium. Furthermore, low sequence similarities of 16S rRNA gene sequences, differences in fatty acid compositions and differential physiological characteristics such as enzyme activity and carbon sources utilization ability distinguished the isolate from its close relatives. Therefore, strain 5221T represents a novel species of the genus Brevibacterium, for which the name Brevibacterium rongguiense sp. nov. is proposed, with the type strain 5221T (=GDMCC 1.1766T=KACC 21700T).

A Molecular Window into the Biology and Epidemiology of Pneumocystis spp.

Pneumocystis, a unique atypical fungus with an elusive lifestyle, has had an important medical history. It came to prominence as an opportunistic pathogen that not only can cause life-threatening pneumonia in patients with HIV infection and other immunodeficiencies but also can colonize the lungs of healthy individuals from a very early age. The genus Pneumocystis includes a group of closely related but heterogeneous organisms that have a worldwide distribution, have been detected in multiple mammalian species, are highly host species specific, inhabit the lungs almost exclusively, and have never convincingly been cultured in vitro, making Pneumocystis a fascinating but difficult-to-study organism. Improved molecular biologic methodologies have opened a new window into the biology and epidemiology of Pneumocystis. Advances include an improved taxonomic classification, identification of an extremely reduced genome and concomitant inability to metabolize and grow independent of the host lungs, insights into its transmission mode, recognition of its widespread colonization in both immunocompetent and immunodeficient hosts, and utilization of strain variation to study drug resistance, epidemiology, and outbreaks of infection among transplant patients. This review summarizes these advances and also identifies some major questions and challenges that need to be addressed to better understand Pneumocystis biology and its relevance to clinical care.

Marmoricola mangrovicus sp. nov., an endophytic actinobacterium isolated from Kandelia candel.

A Gram-stain-positive, aerobic, motile, non-spore-forming and rod-shaped actinobacterium, designated strain 4Q3S-7T, was isolated from a piece of surface-sterilized bark of Kandelia candel collected at the Cotai Ecological Zone in Macao, China. Colonies were yellowish white, circular, smooth and convex. The 16S rRNA gene sequence of strain 4Q3S-7T exhibited highest similarities to Marmoricola ginsengisoli Gsoil 097T (97.6 %), Marmoricola solisilvae KIS18-7T (97.6 %) and Marmoricola pocheonensis Gsoil 818T (97.3 %). Phylogenetic analysis showed that strain 4Q3S-7T clustered with species of the genus Marmoricola and was clearly affiliated to the genus Marmoricola. Genomic analyses, including average nucleotide identity and DNA-DNA hybridization, clearly separated strain 4Q3S-7T from M. ginsengisoli Gsoil 097T, M. solisilvae KIS18-7T and M. pocheonensis Gsoil 818T with values below the thresholds for species delineation. Strain 4Q3S-7T had ll-2,6-diaminopimelic acid as the diagnostic diamino acid in the cell wall. The major fatty acids (>10 % of total fatty acids) were C18 : 0 10-methyl (TBSA), C18 : 1ω9c, iso-C16 : 0 and iso-C16 : 0 2-OH. The predominant menaquinone was MK-8(H4). The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol and an unidentified phospholipid. The DNA G+C content of strain 4Q3S-7T was 72.0 mol% (draft genome sequence). Based on its phylogenetic, phenotypic and chemotaxonomic features, strain 4Q3S-7T is considered to represent a novel species of the genus Marmoricola, for which the name Marmoricola mangrovicus sp. nov. is proposed. The type strain is 4Q3S-7T (=KCTC 39790T=CGMCC 4.7424T).

Large-scale analysis reveals that the genome features of simple sequence repeats are generally conserved at the family level in insects.

BACKGROUND: Simple sequence repeats (SSR), also called microsatellites, have been widely used as genetic markers, and have been extensively studied in some model insects. At present, the genomes of more than 100 insect species are available. However, the features of SSRs in most insect genomes remain largely unknown. RESULTS: We identified 15.01 million SSRs across 136 insect genomes. The number of identified SSRs was positively associated with genome size in insects, but the frequency and density per megabase of genomes were not. Most insect SSRs (56.2-93.1%) were perfect (no mismatch). Imperfect (at least one mismatch) SSRs (average length 22-73 bp) were longer than perfect SSRs (16-30 bp). The most abundant insect SSRs were the di- and trinucleotide types, which accounted for 27.2% and 22.0% of all SSRs, respectively. On average, 59.1%, 36.8%, and 3.7% of insect SSRs were located in intergenic, intronic, and exonic regions, respectively. The percentages of various types of SSRs were similar among insects from the same family. However, they were dissimilar among insects from different families within orders. We carried out a phylogenetic analysis using the SSR frequencies. Species from the same family were generally clustered together in the evolutionary tree. However, insects from the same order but not in the same family did not cluster together. These results indicated that although SSRs undergo rapid expansions and contractions in different populations of the same species, the general genomic features of insect SSRs remain conserved at the family level. CONCLUSION: Millions of insect SSRs were identified and their genome features were analyzed. Most insect SSRs were perfect and were located in intergenic regions. We presented evidence that the variance of insect SSRs accumulated after the differentiation of insect families.

The complete chloroplast genome sequence and phylogenetic analysis of Chuanminshen (Chuanminshenviolaceum Sheh et Shan).

Chloroplast genome sequences are very useful for species identification and phylogenetics. Chuanminshen (Chuanminshen violaceum Sheh et Shan) is an important traditional Chinese medicinal plant, for which the phylogenetic position is still controversial. In this study, the complete chloroplast genome of Chuanminshen violaceum Sheh et Shan was determined. The total size of Chuanminshen chloroplast genome was 154,529 bp with 37.8% GC content. It has the typical quadripartite structure, a large single copy (17,800 bp) and a small single copy (84,171 bp) and a pair of inverted repeats (26,279 bp). The whole genome harbors 132 genes, which includes 85 protein coding genes, 37 tRNA genes, eight rRNA genes, and two pseudogenes. Thirty-nine SSR loci, 32 tandem repeats and 49 dispersed repeats were found. Phylogenetic analyses results with the help of MEGA showed a new insight for the Chuanminshen phylogenetic relationship with the reported chloroplast genomes in Apiales plants.

FEATnotator: A tool for integrated annotation of sequence features and variation, facilitating interpretation in genomics experiments.

As approaches are sought for more efficient and democratized uses of non-model and expanded model genomics references, ease of integration of genomic feature datasets is especially desirable in multidisciplinary research communities. Valuable conclusions are often missed or slowed when researchers refer experimental results to a single reference sequence that lacks integrated pan-genomic and multi-experiment data in accessible formats. Association of genomic positional information, such as results from an expansive variety of next-generation sequencing experiments, with annotated reference features such as genes or predicted protein binding sites, provides the context essential for conclusions and ongoing research. When the experimental system includes polymorphic genomic inputs, rapid calculation of gene structural and protein translational effects of sequence variation from the reference can be invaluable. Here we present FEATnotator, a lightweight, fast and easy to use open source software program that integrates and reports overlap and proximity in genomic information from any user-defined datasets including those from next generation sequencing applications. We illustrate use of the tool by summarizing whole genome sequence variation of a widely used natural isolate of Arabidopsis thaliana in the context of gene models of the reference accession. Previous discovery of a protein coding deletion influencing root development is replicated rapidly. Appropriate even in investigations of a single gene or genic regions such as QTL, comprehensive reports provided by FEATnotator better prepare researchers for interpretation of their experimental results. The tool is available for download at http://featnotator.sourceforge.net.

The first report describes features of the chloroplast genome of Withania frutescens.

Genomic studies not only help researcher not only to identify genomic features in organisms, but also facilitate understanding of evolutionary relationships. Species in the Withania genus have medicinal benefits, and one of them is Withania frutescens, which is used to treat various diseases. This report investigates the nucleotides and genic features of chloroplast genome of Withania frutescens and trying to clarify the evolutionary relationship with Withania sp and family Solanaceae. We found that the total size of Withania frutescens chloroplast genome was 153.771 kb (the smallest chloroplast genome in genus Withania). A large single-copy region (91.285 kb), a small single-copy region (18.373 kb) form the genomic region, and are distinct from each other by a large inverted repeat (22.056 kb). 137 chloroplast genes are found including 4 rRNAs, 38 tRNAs and 83 protein-coding genes. The Withania frutescens chloroplast genome as well as four closest relatives was compared for features such as structure, nucleotide composition, simple sequence repeats (SSRs) and codon bias. Compared to other Withania species, Withania frutescens has unique characteristics. It has the smallest chloroplast genome of any Withania species, isoleucine is the major amino acid, and tryptophan is the minor, In addition, there are no ycf3 and ycf4 genes, fourth, there are only fifteen replicative genes, while in most other species there are more. Using fast minimum evolution and neighbor joining, we have reconstructed the trees to confirm the relationship with other Solanacaea species. The Withania frutescens chloroplast genome is submitted under accession no. ON153173.

Comparative Mitogenomics Reveals Cryptic Species in Sillago ingenuua McKay, 1985 (Perciformes: Sillaginidae).

It is unreliable to identify marine fishes only by external morphological features. Species misidentification brings great challenges to fishery research, resource monitoring and ecomanagement. Sillago ingenuua is an important part of commercial marine fishes, and in which, the morphological differences between different groups are not obvious. Here, we compared different geographical groups of S. ingenuua which were collected from Xiamen, Dongshan, Keelung, Songkhla and Java. The results showed that all samples of S. ingenuua were similar in external morphological characteristics and the shape of the swim bladder, but there were two distinctive lineages which were flagged as cryptic species based on DNA barcoding. The comparative mitogenomic results showed that S. ingenuua A and S. ingenuua B were identical in structural organization and gene arrangement. Their nucleotide composition and codon usage were also similar. A phylogenetic analysis was performed based on 13 concatenated PCGs from eight Sillago species. The results showed that the genetic distance between S. ingenuua A and S. ingenuua B was large (D = 0.069), and this genetic distance was large enough to reveal that S. ingenuua A and S. ingenuua B might be different species.

Monkeypox virus: The changing facets of a zoonotic pathogen.

In the last five years, the prevalence of monkeypox has been increasing both in the regions considered endemic for the disease (West and Central Africa) and worldwide. Indeed, in July 2022, the World Health Organization declared the ongoing global outbreak of monkeypox a public health emergency of international concern. The disease is caused by monkeypox virus (MPXV), a member of the Orthopoxvirus genus, which also includes variola virus (the causative agent of smallpox) and vaccinia virus (used in the smallpox eradication campaign). Here, we review aspects of MPXV genetic diversity and epidemiology, with an emphasis on its genome structure, host range, and relationship with other orthopoxviruses. We also summarize the most recent findings deriving from the sequencing of outbreak MPXV genomes, and we discuss the apparent changing of MPXV evolutionary trajectory, which is characterized by the accumulation of point mutations rather than by gene gains/losses. Whereas the availability of a vaccine, the relatively mild presentation of the disease, and its relatively low transmissibility speak in favor of an efficient control of the global outbreak, the wide host range of MPXV raises concerns about the possible establishment of novel reservoirs. We also call for the deployment of field surveys and genomic surveillance programs to identify and control the MPXV reservoirs in West and Central Africa.

Genome Features of Asaia sp. W12 Isolated from the Mosquito Anopheles stephensi Reveal Symbiotic Traits.

Asaia bacteria commonly comprise part of the microbiome of many mosquito species in the genera Anopheles and Aedes, including important vectors of infectious agents. Their close association with multiple organs and tissues of their mosquito hosts enhances the potential for paratransgenesis for the delivery of antimalaria or antivirus effectors. The molecular mechanisms involved in the interactions between Asaia and mosquito hosts, as well as Asaia and other bacterial members of the mosquito microbiome, remain underexplored. Here, we determined the genome sequence of Asaia strain W12 isolated from Anopheles stephensi mosquitoes, compared it to other Asaia species associated with plants or insects, and investigated the properties of the bacteria relevant to their symbiosis with mosquitoes. The assembled genome of strain W12 had a size of 3.94 MB, the largest among Asaia spp. studied so far. At least 3585 coding sequences were predicted. Insect-associated Asaia carried more glycoside hydrolase (GH)-encoding genes than those isolated from plants, showing their high plant biomass-degrading capacity in the insect gut. W12 had the most predicted regulatory protein components comparatively among the selected Asaia, indicating its capacity to adapt to frequent environmental changes in the mosquito gut. Two complete operons encoding cytochrome bo3-type ubiquinol terminal oxidases (cyoABCD-1 and cyoABCD-2) were found in most Asaia genomes, possibly offering alternative terminal oxidases and allowing the flexible transition of respiratory pathways. Genes involved in the production of 2,3-butandiol and inositol have been found in Asaia sp. W12, possibly contributing to biofilm formation and stress tolerance.

The complete mitochondrial genome of Amphinemura claviloba (Wu, 1973) (Plecoptera: Nemouridae).

We sequenced the complete mitochondrial genome (mitogenome) of a stonefly, Amphinemura claviloba (Wu, 1973), of the family Nemouridae (Insecta: Plecoptera). The mitogenome was 15,707 bp long and contained typical 37 genes with an A+T content of 68.5%. All protein-coding genes (PCGs) use standard initiation codons (methionine and isoleucine), except ND1 and ND5 which starts with TTG and GTG, respectively. Two of the 13 PCGs harbor the incomplete termination codon. All tRNA genes have typical clover secondary structures, except the dihydrouridine (DHU) arm of tRNASer(AGN) forms a simple loop. Secondary structure models of the ribosomal RNA genes of A. claviloba are similar to those proposed for other insects. We also found some structural elements in the control region, such as tandem repeats, poly-C stretch and microsatellite-like elements, etc. Phylogenetic analyses showed the clades for the Nemoura, Amphinemura, and (Mesonemoura + Sphaeronemoura + Indonemoura + Protonemura) are well supported in a polytomy.

Comparative Genomics and Phylogenetic Analyses of Christia vespertilionis and Urariopsis brevissima in the Tribe Desmodieae (Fabaceae: Papilionoideae) Based on Complete Chloroplast Genomes.

Taxonomic and phylogenetic relationships of Christia, Urariopsis, Uraria and related genera within the tribe Desmodieae (Fabaceae: Papilionoideae) have long been controversial. Here, we report the complete chloroplast (cp) genomes of Christia vespertilionis and Urariopsis brevissima and perform comparative and phylogenetic analyses with Uraria lagopodioides and other relatives in the Desmodieae. The cp genomes of C. vespertilionis and U. brevissima are 149,656 and 149,930 bp long, with 128 unique genes (83 protein-coding genes, 37 tRNA genes and 8 rRNA genes), respectively. Comparative analyses revealed 95-129 simple sequence repeats (SSRs) and eleven highly variable regions (trnK-rbcL, rbcL-atpB, ndhJ-trnF, trnL-trnT, psbD-rpoB, accD-cemA, petA-psbL, psbE-petL, rps11-rps19, ndhF-ccsA, and rps15-ycf1) among six Desmodieae species. Phylogenetic analyses clearly resolved two subtribes (Desmodiinae and Lespedezinae) of Desmodieae as monophyletic, and the newly reported C. vespertilionis and U. brevissima clustered in subtribe Desmodiinae. A sister relationship of C. vespertilionis to U. lagopodioides was supported. Evidence was presented to support the treatment of Urariopsis as a distinct genus rather than in synonymy with Uraria. The results provide valuable information for further studies on species delimitation, phylogenetics, population genetics, and the evolutionary process of speciation in the Desmodieae.

The complete chloroplast genome of Colobanthus apetalus (Labill.) Druce: genome organization and comparison with related species.

Colobanthus apetalus is a member of the genus Colobanthus, one of the 86 genera of the large family Caryophyllaceae which groups annual and perennial herbs (rarely shrubs) that are widely distributed around the globe, mainly in the Holarctic. The genus Colobanthus consists of 25 species, including Colobanthus quitensis, an extremophile plant native to the maritime Antarctic. Complete chloroplast (cp) genomes are useful for phylogenetic studies and species identification. In this study, next-generation sequencing (NGS) was used to identify the cp genome of C. apetalus. The complete cp genome of C. apetalus has the length of 151,228 bp, 36.65% GC content, and a quadripartite structure with a large single copy (LSC) of 83,380 bp and a small single copy (SSC) of 17,206 bp separated by inverted repeats (IRs) of 25,321 bp. The cp genome contains 131 genes, including 112 unique genes and 19 genes which are duplicated in the IRs. The group of 112 unique genes features 73 protein-coding genes, 30 tRNA genes, four rRNA genes and five conserved chloroplast open reading frames (ORFs). A total of 12 forward repeats, 10 palindromic repeats, five reverse repeats and three complementary repeats were detected. In addition, a simple sequence repeat (SSR) analysis revealed 41 (mono-, di-, tri-, tetra-, penta- and hexanucleotide) SSRs, most of which were AT-rich. A detailed comparison of C. apetalus and C. quitensis cp genomes revealed identical gene content and order. A phylogenetic tree was built based on the sequences of 76 protein-coding genes that are shared by the eleven sequenced representatives of Caryophyllaceae and C. apetalus, and it revealed that C. apetalus and C. quitensis form a clade that is closely related to Silene species and Agrostemma githago. Moreover, the genus Silene appeared as a polymorphic taxon. The results of this study expand our knowledge about the evolution and molecular biology of Caryophyllaceae.

FLAGdb++: A Bioinformatic Environment to Study and Compare Plant Genomes.

Today, the growing knowledge and data accumulation on plant genomes do not solve in a simple way the task of gene function inference. Because data of different types are coming from various sources, we need to integrate and analyze them to help biologists in this task. We created FLAGdb++ ( http://tools.ips2.u-psud.fr/FLAGdb ) to take up this challenge for a selection of plant genomes. In order to enrich gene function predictions, structural and functional annotations of the genomes are explored to generate meta-data and to compare them. Since data are numerous and complex, we focused on accessibility and visualization with an original and user-friendly interface. In this chapter we present the main tools of FLAGdb++ and a use-case to explore a gene family: structural and functional properties of this family and research of orthologous genes in the other plant genomes.

BEDTools: The Swiss-Army Tool for Genome Feature Analysis.

Technological advances have enabled the use of DNA sequencing as a flexible tool to characterize genetic variation and to measure the activity of diverse cellular phenomena such as gene isoform expression and transcription factor binding. Extracting biological insight from the experiments enabled by these advances demands the analysis of large, multi-dimensional datasets. This unit describes the use of the BEDTools toolkit for the exploration of high-throughput genomics datasets. Several protocols are presented for common genomic analyses, demonstrating how simple BEDTools operations may be combined to create bespoke pipelines addressing complex questions.