The dynamics of genome size and GC contents evolution in genus Nicotiana

Hybridization and Polyploidization are most common of the phenomenon observed in plants, especially in the genus Nicotiana leading to the duplication of genome. Although genomic changes associated with these events has been studied at various levels but the genome size and GC content variation is less understood because of absence of sufficient genomic data. In this study the flow cytometry technique was used to uncover the genome size and GC contents of 46 Nicotiana species and we compared the genomic changes associated with the hybridization events along evolutionary time scale. The genome size among Nicotiana species varied between 3.28 pg and 11.88 pg whereas GC contents varied between 37.22% and 51.25%. The tetraploid species in genus Nicotiana including section Polydiclae, Repandae, Nicotiana, Rustica and Sauveolentes revealed both up and downsizing in their genome sizes when compared to the sum of genomes of their ancestral species. The genome sizes of three homoploid hybrids were found near their ancestral species. Loss of large genome sequence was observed in the evolutionary more aged species (>10 Myr) as compared to the recently evolved one’s (<0.2 Myr). The GC contents were found homogenous with a mean difference of 2.46% among the Nicotiana species. It is concluded that genome size change appeared in either direction whereas the GC contents were found more homogenous in genus Nicotiana.

Genetic and Chemical Diversity of Edible Mushroom Pleurotus Species

The genus Pleurotus is one of the most widely cultivated and edible mushrooms with various cultivators. Three molecular characteristics were used to evaluate the genetic diversity of 132 tested samples. Phylogenetic analysis showed five clades for tested samples of the genus Pleurotus by the combined ITS and LSU sequences with strong bootstraps and Bayesian posterior probability supports. A total of 94 polymorphic fragments ranging from 10 to 100 bp were observed by using an intersimple sequence repeat (ISSR) marker. The DNA fragment pattern showed that P. ostreatus cultivator (strain P9) was clearly distinguished from wild strain based on their clear banding profiles produced. DNA GC content of the genus Pleurotus varied from 55.6 mol% to 43.3 mol%. Their chemical composition was also determined, including sugar, amino acid, polar lipid, mycolic acid, quinone, and fatty acid, which presented some high homogeneity. Most of the tested samples contained mycolic acid; glucose and arabinose as the main sugars; aspartic acid, arginine, lysine, tyrosine, and alanine as the main amino acids; and C16:0, C18:0, C18:2cis-9,12, anteiso-C14:0, and summed feature 8 as the main fatty acids. In addition, their polar lipid profiles were investigated for the first time, which significantly varied among Pleurotus species. The genus Pleurotus contained menaquinone-6 as the sole respiratory quinone, which showed a significant difference with that of its closely related genera. These results of this study demonstrated that the combined method above could efficiently differentiate each Pleurotus species and thus be considered an efficient tool for surveying the genetic diversity of the genus Pleurotus.

S ilencing of SCD and SREBP1 Genes by Short Hairpin RNAs in Chicken Hepatic Cells in Vitro

RNA interference by short hairpin RNAs (shRNAs) is a widely used post transcriptional silencing mechanism for suppressing expression of the target gene. In the current study, five shRNA molecules each against SCD and SREBP1 genes involved in denovo lipid biosynthesis were designed upon considering parameters such as secondary structures of shRNAs, mRNA target regions, GC content and thermodynamic properties (ΔG overall, ΔG duplex and ΔG break-target), synthesized and cloned in pENTR/U6 entry vector to knockdown the expression of SCD and SREBP1 genes. After transfection of these shRNA constructs into the chicken embryonic hepatocytes, expressions of the target genes were monitored by real time PCR. Significant reduction (P<0.05) in the expression of SCD and SREBP1 genes was observed in hepatocytes. The shRNAs against SCD gene showed the knock down efficiency ranged from 20.4% (shRNA5) to 74.2% (shRNA2). In case of SREBP1 gene, the shRNAs showed knock-down efficiency ranging from 26.8% (shRNA4) to 95.85% (shRNA1). The shRNAs against both the genes introduced in chicken hepatocyte cells did not show any significant impact on expression of immune response genes (IFNA and IFNB) in those cells. These results clearly demonstrated the successful down regulation of the expression of SCD and SREBP1 genes by the shRNA molecules against both the target genes under in vitro condition. It is concluded that the shRNA molecules against SCD and SREBP1 genes showed great potential to silence the expression of these genes under in vitro chicken embryonic hepatocyte cells.

Comprehensive profiling of mRNA splicing indicates that GC content signals altered cassette exon inclusion in Ewing sarcoma

ABSTRACT Ewing sarcoma (EwS) is a small round blue cell tumor and is the second most frequent pediatric bone cancer. 85% of EwS tumors express the fusion oncoprotein EWS-FLI1, the product of a t(11;22) reciprocal translocation. Prior work has indicated that transcription regulation alone does not fully describe the oncogenic capacity of EWS-FLI1, nor does it provide an effective means to stratify patient tumors. Research using EwS cell lines and patient samples has suggested that EWS-FLI1 also disrupts mRNA biogenesis. In this work we both describe the underlying characteristics of mRNA that are aberrantly spliced in EwS tumor samples as well as catalogue mRNA splicing events across other pediatric tumor types. Here, we also use short- and long-read sequencing to identify cis-factors that contribute to splicing profiles we observe in Ewing sarcoma. Our analysis suggests that GC content upstream of cassette exons is a defining factor of mRNA splicing in EwS. We also describe specific splicing events that discriminate EwS tumor samples from the assumed cell of origin, human mesenchymal stem cells derived from bone marrow (hMSC-BM). Finally, we identify specific splicing factors PCBP2, RBMX, and SRSF9 by motif enrichment and confirm findings from tumor samples in EwS cell lines.

GC content of plant genes is linked to past gene duplications

The frequency of G and C nucleotides in genomes varies from species to species, and sometimes even between different genes in the same genome. The monocot grasses have a bimodal distribution of genic GC content absent in dicots. We categorized plant genes from 5 dicots and 4 monocot grasses by synteny to related species and determined that syntenic genes have significantly higher GC content than non-syntenic genes at their 5`-end in the third position within codons for all 9 species. Lower GC content is correlated with gene duplication, as lack of synteny to distantly related genomes is associated with past interspersed gene duplications. Two mutation types can account for biased GC content, mutation of methylated C to T and gene conversion from A to G. Gene conversion involves non-reciprocal exchanges between homologous alleles and is not detectable when the alleles are identical or heterozygous for presence-absence variation, both likely situations for genes duplicated to new loci. Gene duplication can cause production of siRNA which can induce targeted methylation, elevating mC→T mutations. Recently duplicated plant genes are more frequently methylated and less likely to undergo gene conversion, each of these factors synergistically creating a mutational environment favoring AT nucleotides. The syntenic genes with high GC content in the grasses compose a subset that have undergone few duplications, or for which duplicate copies were purged by selection. We propose a “biased gene duplication / biased mutation” (BDBM) model that may explain the origin and trajectory of the observed link between duplication and genic GC bias. The BDBM model is supported by empirical data based on joint analyses of 9 angiosperm species with their genes categorized by duplication status, GC content, methylation levels and functional classes.

Complete Genome Sequence of Curtobacterium sp. Strain TXMA1, Isolated from a Grapevine in Texas, USA

The genome of Curtobacterium sp. strain TXMA1, isolated from a grapevine in Texas showing leaf marginal necrosis symptoms, was sequenced. The TXMA1 genome has a 3,454,876-bp, circular chromosome with a GC content of 71.74%, 3,213 open reading frames (ORFs), 47 tRNAs, and 4 complete rRNA operons (5S, 16S, and 23S).

Whole Genome Sequencing Assembly and Annotation of Halobacillus Trueperi S61 Isolated From the Qarhan Salt Lake

Background: Halophilic microbial as prospective resources of biotechnology due to the advantages of flexible survivability. Qarhan Salt Lake is the second largest Salt Lake in the world which contains rich-unique extremophiles and deserved in-depth exploration. Results: Present study first time isolated novel strain Halobacillus trueperi S61 from Qarhan Salt Lake and performed whole-genome sequencing through combined third-generation PacBio and second-generation Illumina technology. The whole genome of Halobacillus trueperi S61 identified 57549 total reads and consists a complete circular chromosome of 4047887 bp with 43.86% GC content without gaps. Total number of 139 non-coding RNA (included 86 tRNA, 30 rRNA and 23 sRNA), 16 gene islands with 260275 bp and two prophages (with 82682 length) were predicted. In addition, the whole genome of Halobacillus trueperi S61 summarized basic annotation for 3982 protein-coding genes, 3980, 3667, 2998 and 2303 unigenes were annotated with Nr, Swissport, KOG and KEGG database. Combined with advanced analysis, 561 carbohydrate enzymes and 4416 pathogen host interactions related genes were identified. The protein function of Halobacillus trueperi S61 was mainly focus on biological processes, and the protein function was mainly distributed in gene transcription and amino acids, and carbohydrates metabolism. Conclusions: The complete whole genome sequence assembly and annotation of novel strain Halobacillus trueperi S61 isolated from Qarhan Salt Lake mainly focus on protein biological processes and antibiotic resistance, provides a potential resource for biotechnology.

The Complete Chloroplast Genome of Ardisia Japonica（Thunb.）Blume

Ardisia japonica（Thunb）Blume is a small shrub or sub-shrub of the genus Taurus in the Taurus family. The whole plant and root of A. japonica are used for medicinal purposes. It is a common Chinese herbal medicine and a common flower. To study its complete chloroplast genome, we collected leaves and obtained chloroplast genome information through next-generation sequencing. The results showed that the length of the genome is 155,996 bp, and the GC content ratio is 37.0%. The large single-copy region (LSC) is 86803 bp, the small single-copy region (SSC) is 18080 bp, and the inverted repeat region (IR) is 25507 bp. The chloroplast genome encodes 130 genes, including 85 protein genes, 8 rRNA genes, and 37tRNA genes. By analyzing the phylogeny of A. japonica, it is found that A. japonica and other Ardisia species are closely related.

SGP-C: A Broad Host Range Temperate Bacteriophage; Against Salmonella gallinarum

Salmonella gallinarum is a poultry restricted-pathogen causing fowl-typhoid disease in adult birds with mortality rates up-to 80% and exhibit resistance against commonly used antibiotics. In this current study, a temperate broad host range bacteriophage SGP-C was isolated against S. gallinarum from poultry digesta. It showed infection ability in all the 15 tested field strains of S. gallinarum. The SGP-C phage produced circular, turbid plaques with alternate rings. Its optimum activity was observed at pH 7.0 and 37–42°C, with a latent period of 45 min and burst size of 187 virions/bacterial cell. The SGP-C lysogens, SGPC-L5 and SGPC-L6 exhibited super-infection immunity against the same phage, an already reported feature of lysogens. A virulence index of 0.5 and 0.001 as MV50 of SGP-C suggests its moderate virulence. The genome of SGP-C found circular double stranded DNA of 42 Kbp with 50.04% GC content, which encodes 63 ORFs. The presence of repressor gene at ORF49, and absence of tRNA sequence in SGP-C genome indicates its lysogenic nature. Furthermore, from NGS analysis of lysogens we propose that SGP-C genome might exist either as an episome, or both as integrated and temporary episome in the host cell and warrants further studies. Phylogenetic analysis revealed its similarity with Salmonella temperate phages belonging to family Siphoviridae. The encoded proteins by SGP-C genome have not showed homology with any known toxin and virulence factor. Although plenty of lytic bacteriophages against this pathogen are already reported, to our knowledge SGP-C is the first lysogenic phage against S. gallinarum reported so far.

GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria. We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2–79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 105 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45–77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria, representing a candidate order (Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O2, suggesting a microaerophilic lifestyle.