Low GC Research Articles

Glucocorticoids in preterm human milk.

BackgroundGlucocorticoids (GCs), cortisol and cortisone, are essential regulators of many physiological responses, including immunity, stress and mammary gland function. GCs are present in human milk (HM), but whether maternal and infant factors are associated with HM GC concentration following preterm birth is unclear.Materials and methodsHM samples were collected on postnatal day 5 and 10 and at 4 months’ corrected age (4m CA) in a cohort of moderate- and late-preterm infants. GCs in HM were measured by liquid chromatography-tandem mass spectrometry. Relationships between GCs in HM and both maternal and infant characteristics were investigated using Spearman’s correlations and linear mixed models.Results170 mothers of 191 infants provided 354 HM samples. Cortisol concentrations in HM increased from postnatal day 5–4m CA (mean difference [MD] 0.6 ± 0.1 ng/ml, p < 0.001). Cortisone concentration did not change across lactation but was higher than cortisol throughout. Compared to no antenatal corticosteroid (ANS), a complete course of ANS was associated with lower GC concentrations in HM through to 4m CA (cortisol: MD –0.3 ± 0.1 ng/ml, p < 0.01; cortisone MD –1.8 ± 0.4 ng/ml, p < 0.001). At 4m CA, higher maternal perceived stress was negatively associated with GC concentrations in HM (cortisol adjusted beta-coefficient [aβ] –0.01 ± 0.01 ng/ml, p = 0.05; and cortisone aβ –0.1 ± 0.03 ng/ml, p = 0.01), whereas higher postpartum depression and maternal obesity were associated with lower cortisone concentrations (aβ –0.1 ± 0.04 ng/ml p < 0.05; MD [healthy versus obese] –0.1 ± 0.04 ng/ml p < 0.05, respectively). There was a weak positive correlation between GC concentrations in HM and gestational age at birth (r = 0.1, p < 0.05). Infant birth head circumference z-score was negatively associated with cortisol concentrations (aβ –0.01 ± 0.04 ng/ml, p < 0.05). At hospital discharge, fat-free mass showed a weak positive correlation with cortisol concentrations (r = 0.2, p = 0.03), while fat mass showed a weak negative correlation with cortisone concentrations (r = –0.25, p < 0.001).ConclusionThe mammary gland appears to protect the infant from cortisol through inactivation into cortisone. Maternal and infant characteristics were associated with concentration of GCs in HM, including ANS, stress and depression scores, obesity, gestational age and infant size. The effects of HM glucocorticoids on long-term health outcomes requires further research.

The extremely reduced, diverged and reconfigured plastomes of the largest mycoheterotrophic orchid lineage

BackgroundPlastomes of heterotrophic plants have been greatly altered in structure and gene content, owing to the relaxation of selection on photosynthesis-related genes. The orchid tribe Gastrodieae is the largest and probably the oldest mycoheterotrophic clade of the extant family Orchidaceae. To characterize plastome evolution across members of this key important mycoheterotrophic lineage, we sequenced and analyzed the plastomes of eleven Gastrodieae members, including representative species of two genera, as well as members of the sister group Nervilieae.ResultsThe plastomes of Gastrodieae members contain 20 protein-coding, four rRNA and five tRNA genes. Evolutionary analysis indicated that all rrn genes were transferred laterally and together, forming an rrn block in the plastomes of Gastrodieae. The plastome GC content of Gastrodia species ranged from 23.10% (G. flexistyla) to 25.79% (G. javanica). The plastome of Didymoplexis pallens contains two copies each of ycf1 and ycf2. The synonymous and nonsynonymous substitution rates were very high in the plastomes of Gastrodieae among mycoheterotrophic species in Orchidaceae and varied between genes.ConclusionsThe plastomes of Gastrodieae are greatly reduced and characterized by low GC content, rrn block formation, lineage-specific reconfiguration and gene content, which might be positively selected. Overall, the plastomes of Gastrodieae not only serve as an excellent model for illustrating the evolution of plastomes but also provide new insights into plastome evolution in parasitic plants.

The divergent genome of Scorpion Group 1 (SG1) intracellular bacterium from the venom glands of Vaejovis smithi (Scorpiones: Vaejovidae)

Scorpions were among the first animals on land around 430 million years ago. Like many arachnids, scorpions have evolved complex venoms used to paralyze their prey and for self-defense. Here we sequenced and analyzed the metagenomic DNA from venom glands from Vaejovis smithi scorpions. A metagenome-assembled genome (MAG) of 624,025 bp was obtained corresponding to the previously reported Scorpion Group 1 (SG1). The SG1 genome from venom glands had a low GC content (25.8%) characteristic of reduced genomes, many hypothetical genes and genes from the reported minimal set of bacterial genes. Phylogenomic reconstructions placed the uncultured SG1 distant from other reported bacteria constituting a taxonomic novelty. By PCR we detected SG1 in all tested venom glands from 30 independent individuals. Microscopically, we observed SG1 inside epithelial cells from the venom glands using FISH and its presence in scorpion embryos suggested that SG1 is transferred from mother to offspring.

Comparative Genomics of Six Lytic Bacillus subtilis Phages from the Southwest United States.

Despite their importance to microbial dynamics involving Bacillus subtilis, we have a limited understanding of the diversity of phages that can lyse this model organism. Phages were isolated from soil samples collected from various sites in the southwest U.S. deserts on a wild B. subtilis strain. Their genomes were assembled, characterized, and bioinformatically compared. Six Siphoviruses with high nucleotide and amino acid similarity to each other (>80%) but very limited similarity to phages currently in GenBank were isolated. These phages have double-stranded DNA genomes (55,312 to 56,127 bp) with 86-91 putative protein coding genes, and a low GC content. Comparative genomics reveal differences in loci encoding proteins that are putatively involved in bacterial adsorption with evidence for genomic mosaicism and a possible role for small genes. A comparative approach provides insights into phage evolution, including the role of indels in protein folding.

Highly Contiguous Genome Assemblies of the Guinea Paper Wasp (Polistes exclamans) and Mischocyttarus mexicanus.

Paper wasps are a model system for the study of social evolution due to a high degree of inter- and intraspecific variation in cooperation, aggression, and visual signals of social status. Increasing the taxonomic coverage of genomic resources for this diverse clade will aid comparative genomic approaches for testing predictions about the molecular basis of social evolution. Here, we provide draft genome assemblies for two well-studied species of paper wasps, Polistes exclamans and Mischocyttarus mexicanus. The P. exclamans genome assembly is 221.5 Mb in length with a scaffold N50 of 4.11 Mb. The M. mexicanus genome assembly is 227 Mb in length with a scaffold N50 of 1.1 Mb. Genomes have low repeat content (9.54–10.75%) and low GC content (32.06–32.4%), typical of other social hymenopteran genomes. The DNA methyltransferase gene, Dnmt3 , was lost early in the evolution of Polistinae. We identified a second independent loss of Dnmt3 within hornets (genus: Vespa).

Genetic variation and evolution of attenuated African swine fever virus strain isolated in the field

It has been reported that there were several "mutant isolated in the field " of African swine fever virus (ASFV) since ASFV was reported, which may be the result of the continuous adaptation and evolution of ASFV. The emergence of ASFV field mutants may lead to chronic or asymptomatic "atypical clinical symptoms" in pigs and hinder the development of porcine industry. Here we analyzed the published ASFV "field attenuated strain" gene sequences and reviewed the genetic differences between field attenuated and virulent ASFV strains, hoping for providing a reference for the scientific prevention and control of ASF and the development of new vaccines. In this study we found the deletion of EP153R and EP402R occurred in 4 field attenuated strains, and all the differential genes of field attenuated strains mainly range in regions with low GC content. The evolution of MGF110 family genes was identified by analysis of two field attenuated ASFV strains from Portugal. We also found that some tandem repeat sequence plays an important role in the evolution of strains of NH/P68 and OURT 88/3 but not in strains Estonia 2014, HuB20 and Pig/Heilongjiang/HRB1/2020.

Whole human genome 5’-mC methylation analysis using long read nanopore sequencing

ABSTRACT Methylation microarray and bisulphite sequencing are often used to study 5’-methylcytosine (5’-mC) modification of CpG dinucleotides in the human genome. Although both technologies produce trustworthy results, the evaluation of the methylation status of CpG sites suffers from the potential side effects of DNA modification by bisulphite and/or the ambiguity of mapping short reads in repetitive and highly homologous genomic regions, respectively. Nanopore sequencing is an attractive alternative for the study of 5’-mC since it allows sequencing of native DNA molecules, whereas the long reads produced by this technology help to increase the resolution of those genomic regions. In this work, we show that nanopore sequencing with 10X coverage depth, using DNA from a human cell line, produces 5’-mC methylation frequencies consistent with those obtained by 450k microarray, digital restriction enzyme analysis of methylation, and reduced representation bisulphite sequencing. High correlation between methylation frequencies obtained by nanopore sequencing and the other methodologies was also noticeable in either low or high GC content regions, including CpG islands and transcription start sites. We also showed that a minimum of five reads per CpG yields strong correlations (>0.89) in replicate nanopore sequencing runs and an almost uniform linearity of the methylation frequency variation between zero and one. Furthermore, nanopore sequencing was able to correctly display methylation frequency patterns based on genomic annotations of CpG regions. These results demonstrate that nanopore sequencing is a fast, robust, and reliable approach to the study of 5’-mC in the human genome with low coverage depth.

Sustainability by Function (SbF): A Case Study in a Rainfed Vineyard to Reduce the Loss of Soil Nutrients

The effectiveness of a seeded cover crop to minimize soil nutrient losses was evaluated in a rainfed vineyard. Two sediment tanks were installed (ST2: drainage area with high ground cover (GC: 82%) and ST3: very high GC (89%)) and samples from 26 time-integrated periods (TIP) were collected over 15 months. The average soil nutrient content was previously estimated in the drainage areas of ST2 (Ntotal: 0.967 mg/g; Pava: 0.411 mg/g; Kava: 1.762 mg/g) and ST3 (Ntotal: 0.711 mg/g; Pava: 0.437 mg/g; Kava: 1.856 mg/g). The sediment nutrient concentrations and the sediment/soil enrichment ratios were comparable between ST2 and ST3, but the total loss of nutrients clearly differed among areas. The loss of nutrients in the area with lower GC (379.7 g N-P-K/ha/yr) was 8.3 times higher than in the area with higher GC (45.8 g N-P-K/ha/yr), and this pattern remained during the months with low, medium and high GC: 91.9, 2.1 and 2.1 g N-P-K/ha/month in ST2 and 6.9, 3.0 and 3.5 g N-P-K/ha/month in ST3. The benefits of greater GC promote the environmental and agronomic sustainability by the functions of the cover crop, favoring healthy soils and a reduction in the investment of the farmers in fertilizers. This is very relevant in a postpandemic world under the threat of the war in Ukraine, the lack of fertilizers and the need for a local production of food.

The evolution of extremely diverged plastomes in Selaginellaceae (lycophyte) is driven by repeat patterns and the underlying DNA maintenance machinery.

Two factors are proposed to account for the unusual features of organellar genomes: the disruptions of organelle-targeted DNA replication, repair, and recombination (DNA-RRR) systems in the nuclear genome and repetitive elements in organellar genomes. Little is known about how these factors affect organellar genome evolution. The deep-branching vascular plant family Selaginellaceae is known to have a deficient DNA-RRR system and convergently evolved organellar genomes. However, we found that the plastid genome (plastome) of Selaginella sinensis has extremely accelerated substitution rates, a low GC content, pervasive repeat elements, a dynamic network structure, and it lacks direct or inverted repeats. Unexpectedly, its organelle DNA-RRR system is short of a plastid-targeted Recombinase A1 (RecA1) and a mitochondrion-targeted RecA3, in line with other explored Selaginella species. The plastome contains a large collection of short- and medium-sized repeats. Given the absence of RecA1 surveillance, we propose that these repeats trigger illegitimate recombination, accelerated mutation rates, and structural instability. The correlations between repeat quantity and architectural complexity in the Selaginella plastomes support these conclusions. We, therefore, hypothesize that the interplay of the deficient DNA-RRR system and the high repeat content has led to the extraordinary divergence of the S. sinensis plastome. Our study not only sheds new light on the mechanism of plastome divergence by emphasizing the power of cytonuclear integration, but it also reconciles the longstanding contradiction on the effects of DNA-RRR system disruption on genome structure evolution.

Mining transcriptome data: Utilization of environmentally regulated promoters for protein expression and purification in Clostridium perfringens

Clostridium perfringens is a Gram-positive pathogen with low GC content. To identify genes that are transcribed at higher levels when the bacteria grow on a surface, we used RNA-seq in a previous study to measure global transcript levels of cells grown in three types of media on both plates and in liquid culture. We found the arcABDC-argR operon is induced >1000-fold when the cells were grown on plates than in liquid brain heart infusion (BHI). In addition, the pyrBICFZDE operon was transcribed >1000-fold higher in liquid BHI than on plates. Biochemical analysis of C. perfringens proteins is usually accomplished by cloning and expressing the relevant genes in Escherichia coli, a Gram-negative bacterium. Here we utilize both the arcA and pyrB promoters to express and purify proteins from C. perfringens plate and liquid-grown cultures, respectively. Three mg of the His-tagged cytoplasmic protein PilM were obtained when the pilM gene was expressed in cells grown on 10 BHI plates using the arcA promoter. Using the pyrB promoter, 0.85 mg of the C. perfringens His-tagged secreted toxin collagenase was purified from the culture supernatant of 500 ml of cells grown in liquid BHI. In the process of constructing clones, we found we can transform C. perfringens strain HN13 directly with DNA from an in vitro ligation mix, bypassing E. coli. These environmentally regulated promoters can be used to express clostridial or other low GC content genes for protein purification without the addition of an inducer molecule.

Abstract 3739: EM-seq permits accurate and precise methylome analysis of cfDNA and FFPE DNA

Abstract The study of DNA methylation from cell-free DNA (cfDNA) and DNA extracted from formalin fixed paraffin embedded (FFPE) tissues are notoriously difficult. The use of these DNA inputs is becoming increasingly important in the field of cancer diagnostics. The cytosine modifications, 5-methylcytosines (5mC) and 5-hydroxymethylcytosines (5hmC), are key epigenetic factors that play an important role in cellular processes and their mis-regulation results in diseased states like cancer. Advances in the study of these epigenetic factors in combination with improvements in sample preparation have enabled cancer biomarker identification based on methylation profiling. The historical gold standard for detecting cytosine methylation has been bisulfite sequencing. This method uses chemical conversion of cytosines into uracils and has been used for both targeted and whole genome methylation analysis. However, bisulfite conversion leads to DNA damage which results in shorter DNA insert sizes as well as biases in the data. It is challenging to make bisulfite libraries from cfDNA and FFPE DNA as typically the DNA is of low quantity and quality. We developed an enzyme-based methylation detection technology (EM-seq) to address the flaws inherent to bisulfite sequencing. This method, primarily due to the intact nature of DNA after conversion, results in better quality libraries with longer insert sizes, lower duplication rates and minimal GC bias. EM-seq libraries prepared from cfDNA and lung FFPE DNA had longer inserts, lower duplication rates, higher percentages of mapped reads and less GC bias compared to bisulfite libraries. EM-seq libraries also identified a higher number of CpGs with even coverage facilitating accurate methylation calling. EM-seq’s superior sequencing metrics establish it as a standard method for robust methylation profiling, particularly for these types of challenging DNA samples. Citation Format: Louise Williams. EM-seq permits accurate and precise methylome analysis of cfDNA and FFPE DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3739.

Site quality and intensive early stand management practices affect growth dominance, structural complexity, and tree growth in ponderosa pine plantations

During stand initiation, the influences of silvicultural practices such as competing vegetation control and fertilization have been primarily examined in terms of stand-level growth rather than structural complexity. Understanding how intensive silvicultural treatments alter the long-term trajectory of stand development may be critical to meeting management objectives, such as habitat restoration, minimizing ladder fuels, or carbon sequestration. Previous studies have found mixed evidence for an effect of vegetation control and fertilization on structural complexity. Investigating how early pulse treatments drive tree size differentiation in young stands, as indicated by growth dominance (GD), could potentially allow managers to anticipate or design treatments to alter the development of structural complexity later in stand development. We reanalyzed data from the Garden of Eden experiment, a full-factorial study investigating how competing vegetation and nutrients impact ponderosa pine growth and yield over a gradient of site quality in northern California, USA. Our goals were to determine whether these silvicultural practices alter GD and structural complexity, and whether GD and structural complexity created potential feedbacks to alter stand productivity. We examined data for the first 20 years of stand development across 6 study sites. For each site and observation period, we calculated the growth dominance coefficient of total cubic volume to represent GD and the Gini coefficient of inequality in basal area distribution (GC) to represent structural complexity. We created a series of competing generalized additive mixed models (GAMMs) to disentangle the roles of site, stand age, and treatment on GD and GC. We created a set of competing individual-tree GAMMs at age 20 to isolate the influences of GC and GD on volume increment, controlling for the effects of competition, tree size, site, and treatment influences. In agreement with previous research, we found that GD deviated little from size-symmetry (tree growth being roughly proportional to tree size) and did not vary consistently with treatment. Competing vegetation control and higher site quality promoted lower GC, indicative of lower structural complexity. Fertilization increased structural complexity relative to other treatments on two sites in particular, which may reflect unintentional increases in drought stress from fertilization on sites with metasedimentary soils. While GD had limited influence on individual-tree growth, GC interacted with tree size such that small-medium sized trees grew more rapidly on structurally simple plots. Our results have implications for balancing objectives such as fire risk, carbon sequestration, and habitat during reforestation projects.

Genomic insights into positive selection during barley domestication

BackgroundCultivated barley (Hordeum vulgare) is widely used in animal feed, beverages, and foods and has become a model crop for molecular evolutionary studies. Few studies have examined the evolutionary fates of different types of genes in barley during the domestication process.ResultsThe rates of nonsynonymous substitution (Ka) to synonymous substitution (Ks) were calculated by comparing orthologous genes in different barley groups (wild vs. landrace and landrace vs. improved cultivar). The rates of evolution, properties, expression patterns, and diversity of positively selected genes (PSGs) and negatively selected genes (NSGs) were compared. PSGs evolved more rapidly, possessed fewer exons, and had lower GC content than NSGs; they were also shorter and had shorter intron, exon, and first exon lengths. Expression levels were lower, the tissue specificity of expression was higher, and codon usage bias was weaker for PSGs than for NSGs. Nucleotide diversity analysis revealed that PSGs have undergone a more severe genetic bottleneck than NSGs. Several candidate PSGs were involved in plant growth and development, which might make them as excellent targets for the molecular breeding of barley.ConclusionsOur comprehensive analysis of the evolutionary, structural, and functional divergence between PSGs and NSGs in barley provides new insight into the evolutionary trajectory of barley during domestication. Our findings also aid future functional studies of PSGs in barley.

Proof of concept for multiplex amplicon sequencing for mutation identification using the MinION nanopore sequencer

Rapid, cost-effective identification of genetic variants in small candidate genomic regions remains a challenge, particularly for less well equipped or lower throughput laboratories. The application of Oxford Nanopore Technologies’ MinION sequencer has the potential to fulfil this requirement. We demonstrate a proof of concept for a multiplexing assay that pools PCR amplicons for MinION sequencing to enable sequencing of multiple templates from multiple individuals, which could be applied to gene-targeted diagnostics. A combined strategy of barcoding and sample pooling was developed for simultaneous multiplex MinION sequencing of 100 PCR amplicons. The amplicons are family-specific, spanning a total of 30 loci in DNA isolated from 82 human neurodevelopmental cases and family members. The target regions were chosen for further interrogation because a potentially disease-causative variant had been identified in affected individuals following Illumina exome sequencing. The pooled MinION sequences were deconvoluted by aligning to custom references using the minimap2 aligner software. Our multiplexing approach produced an interpretable and expected sequence from 29 of the 30 targeted genetic loci. The sequence variant which was not correctly resolved in the MinION sequence was adjacent to a five nucleotide homopolymer. It is already known that homopolymers present a resolution problem with the MinION approach. Interestingly despite equimolar quantities of PCR amplicon pooled for sequencing, significant variation in the depth of coverage (127×–19,626×; mean = 8321×, std err = 452.99) was observed. We observed independent relationships between depth of coverage and target length, and depth of coverage and GC content. These relationships demonstrate biases of the MinION sequencer for longer templates and those with lower GC content. We demonstrate an efficient approach for variant discovery or confirmation from short DNA templates using the MinION sequencing device. With less than 130 × depth of coverage required for accurate genotyping, the methodology described here allows for rapid highly multiplexed targeted sequencing of large numbers of samples in a minimally equipped laboratory with a potential cost as much 200 × less than that from Sanger sequencing.

MP05-16 NEW PERSPECTIVES ON AN OLD GROUPING: THE GENOMIC AND PHENOTYPIC DIFFERENCES OF OXALOBACTER FORMIGENES

You have accessJournal of UrologyCME1 May 2022MP05-16 NEW PERSPECTIVES ON AN OLD GROUPING: THE GENOMIC AND PHENOTYPIC DIFFERENCES OF OXALOBACTER FORMIGENES John Chmiel, Robertson Venema, Charles Carr, Kait Al, Ryan Chanyi, Sergio Ari Domínguez Romero, Gerrit Stuivenberg, Hassan Razvi, Jennifer Bjazevic, and Jeremy Burton John ChmielJohn Chmiel More articles by this author , Robertson VenemaRobertson Venema More articles by this author , Charles CarrCharles Carr More articles by this author , Kait AlKait Al More articles by this author , Ryan ChanyiRyan Chanyi More articles by this author , Sergio Ari Domínguez RomeroSergio Ari Domínguez Romero More articles by this author , Gerrit StuivenbergGerrit Stuivenberg More articles by this author , Hassan RazviHassan Razvi More articles by this author , Jennifer BjazevicJennifer Bjazevic More articles by this author , and Jeremy BurtonJeremy Burton More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002522.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Oxalobacter formigenes is a near-ubiquitous bacterium with the unique ability to metabolize oxalate as a sole carbon source. Due to this metabolic function, O. formigenes has a historically complex relationship with stone disease. However, because this organism is known to colonize both stone formers and healthy controls, its role in mitigating stone disease is uncertain. Based on genomic and phenotypic analyses, O. formigenes has been divided into two groups. Oral supplementation to mitigate stone formation has only been tested with group 1 (G1) strains and has demonstrated mixed results. The differences between G1 and group 2 (G2) strains remain unclear. Elucidating these distinctions could provide a better understanding of the physiology and potential clinical applications of the bacterium. METHODS: By combining computational genomics with culture-dependent techniques, we sought to characterize the group differences that exist between strains of O. formigenes. Whole genomes isolated from multiple strains of O. formigenes were sequenced and combined with existing genome assemblies to functionally characterize the strains. In vitro growth curves were used to determine the ability of these strains to use oxalate individually. To investigate how Oxalobacter functions in a diverse microbial setting, a human fecal sample was inoculated into media with or without oxalate and the bacterial community dynamics were monitored over time via 16S rRNA gene sequencing. RESULTS: Phylogenetic analysis confirmed a high degree of intergroup dissimilarity between G1 and G2 strains. G1 strains were found to have a lower GC content, larger genomes, more functional genes, and increased intragroup similarity when compared to the G2 strains. Interestingly, no genes were identified as unique to animal strains. G1 strains grew faster and to higher cell density than G2 strains. Oxalobacter abundance did not significantly increase in the oxalate fecal bacterial consortium over time, but the community composition significantly changed. CONCLUSIONS: These findings highlight that G1 strains are highly related and may have an enhanced capability to degrade oxalate. With respect to survival and colonization, the diversity of G2 strains could provide increased adaptability, a benefit to potential future G2-based therapeutics such as oral supplementation to stone-formers. Additionally, the lack of major differences between human and animal strains indicates that non-human isolates should also be considered as a candidate intervention for stone disease. Source of Funding: CIHR CGS-D & CUA award © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e74 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information John Chmiel More articles by this author Robertson Venema More articles by this author Charles Carr More articles by this author Kait Al More articles by this author Ryan Chanyi More articles by this author Sergio Ari Domínguez Romero More articles by this author Gerrit Stuivenberg More articles by this author Hassan Razvi More articles by this author Jennifer Bjazevic More articles by this author Jeremy Burton More articles by this author Expand All Advertisement PDF DownloadLoading ...

Comparative Analyses of Chloroplast Genomes Provide Comprehensive Insights into the Adaptive Evolution of Paphiopedilum (Orchidaceae)

An elucidation of how the selection pressures caused by habitat environments affect plant plastid genomes and lead to the adaptive evolution of plants, is a very intense area of research in evolutionary biology. The genus Paphiopedilum is a predominant group of orchids that includes over 66 species with high horticultural and ornamental value. However, owing to the destructive exploitation and habitat deterioration of wild germplasm resources of Paphiopedilum, it needs more molecular genetic resources and studies on this genus. The chloroplast is cytoplasmically inherited and often used in evolutionary studies. Thus, for this study, we newly sequenced, assembled and annotated five chloroplast genomes of the Paphiopedilum species. The size of these genomes ranged from 155,886 bp (P. henryanum) to 160,503 bp (P. ‘GZSLKY’ Youyou) and they contained 121–122 genes, which consisted of 76 protein coding genes, eight ribosomal RNAs, and 37–38 transfer RNAs. Combined with the other 14 Paphiopedilum species, the characteristics of the repeat sequences, divergent hotspot regions, and the condo usage bias were evaluated and identified, respectively. The gene transfer analysis showed that some fragments of the ndh and ycf gene families were shared by both the chloroplast and nucleus. Although the genomic structure and gene content was conserved, there was a significant boundary shift caused by the inverted repeat (IR) expansion and small single copy (SSC) contraction. The lower GC content and loss of ndh genes could be the result of adaptive evolutionary responses to its unique habitats. The genes under positive selection, including accD, matK, psbM, rpl20, rps12, ycf1, and ycf2 might be regarded as potential candidate genes for further study, which significantly contribute to the adaptive evolution of Paphiopedilum.

Identification of long non-coding RNAs in Verticillium dahliae following inoculation of cotton

Long non-coding RNAs (lncRNAs) play important roles in diverse biological processes. However, these functions have not been assessed in Verticillium dahliae, a soil-borne fungal pathogen that causes devastating wilt diseases in many crops. The discovery and identity of novel lncRNAs and their association with virulence may contribute to an increased understanding of the regulation of virulence in V. dahliae. Here, we identified a total of 352 lncRNAs in V. dahliae. The lncRNAs were transcribed from all V. dahliae chromosomes, typically with shorter open reading frames, lower GC content, and fewer exons than protein-coding genes. In addition, 308 protein-coding genes located within 10 kb upstream and 10 kb downstream of lncRNAs were identified as neighboring genes, and which were considered as potential targets of lncRNA. These neighboring genes encode products involved in development, stress responses, and pathogenicity of V. dahliae, such as transcription factors (TF), kinase, and members of the secretome. Furthermore, 47 lncRNAs were significantly differentially expressed in V. dahliae following inoculation of susceptible cotton (Gossyoiumhisutum) cultivar Junmian No.1, suggesting that lncRNAs may be involved in the regulation of virulence in V. dahliae. Moreover, correlations in expression patterns between lncRNA and their neighboring genes were detected. Expression of lncRNA012077 and its neighboring gene was up-regulated 6 h following inoculation of cotton, while the expression of lncRNA007722 was down-regulated at 6 h but up-regulated at 24 h, in a pattern opposite to that of its neighboring gene. Overexpression of lncRNA012077 in wild-type strain (Vd991) enhanced its virulence on cotton while overexpression of lncRNA009491 reduced virulence. Identification of novel lncRNAs and their association with virulence may provide new targets for disease control.

GC content strongly influences the role of poly(dA) in the intrinsic nucleosome positioning in Saccharomyces cerevisiae.

The nucleosome is the basic structural element of genomic DNA packaging and plays a role in transcription, replication, and recombination. Poly(dA) tracts are considered major sequence determinants of nucleosome positioning, although their role is not well understood. Here, we show that the homopolymeric character and the low GC content of poly(dA)s play different roles in nucleosome formation. We found that the inherent low GC content of poly(dA) alone can account for the deep and anisotropic nucleosome depletion at structurally and functionally important regions of promoters and origins of replication. We also show that the level of nucleosome occupancy at poly(dA) is strongly related to the local nucleotide background and its high frequency of occurrence in Saccharomyces cerevisiae does not appear merely to be associated with its intrinsic nucleosome‐excluding properties. In addition, we show that the GC content alone can predict more than 60% of the in vitro nucleosome map, providing further evidence that the intrinsic nucleosome positioning is more greatly determined by GC content than poly(dA) stretches. Our results are consistent with a model in which poly(dA) stretches act at two distinct levels: first, by its low GC content, which intrinsically contributes to hinder nucleosome formation, and second, by its contiguous runs of dA that selectively drive the recruitment of non‐histone proteins with structural and functional roles.

Genome-wide identification and characterization of Fusarium circinatum-responsive lncRNAs in Pinus radiata

BackgroundOne of the most promising strategies of Pine Pitch Canker (PPC) management is the use of reproductive plant material resistant to the disease. Understanding the complexity of plant transcriptome that underlies the defence to the causal agent Fusarium circinatum, would greatly facilitate the development of an accurate breeding program. Long non-coding RNAs (lncRNAs) are emerging as important transcriptional regulators under biotic stresses in plants. However, to date, characterization of lncRNAs in conifer trees has not been reported. In this study, transcriptomic identification of lncRNAs was carried out using strand-specific paired-end RNA sequencing, from Pinus radiata samples inoculated with F. circinatum at an early stage of infection.ResultsOverall, 13,312 lncRNAs were predicted through a bioinformatics approach, including long intergenic non-coding RNAs (92.3%), antisense lncRNAs (3.3%) and intronic lncRNAs (2.9%). Compared with protein-coding RNAs, pine lncRNAs are shorter, have lower expression, lower GC content and harbour fewer and shorter exons. A total of 164 differentially expressed (DE) lncRNAs were identified in response to F. circinatum infection in the inoculated versus mock-inoculated P. radiata seedlings. The predicted cis-regulated target genes of these pathogen-responsive lncRNAs were related to defence mechanisms such as kinase activity, phytohormone regulation, and cell wall reinforcement. Co-expression network analysis of DE lncRNAs, DE protein-coding RNAs and lncRNA target genes also indicated a potential network regulating pectinesterase activity and cell wall remodelling.ConclusionsThis study presents the first comprehensive genome-wide analysis of P. radiata lncRNAs and provides the basis for future functional characterizations of lncRNAs in relation to pine defence responses against F. circinatum.

GC content, but not nucleosome positioning, directly contributes to intron splicing efficiency in Paramecium.

Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the more than 90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur cotranscriptionally, and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We show that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of ∼151 bp, among the smallest periodicities reported. Our analysis has revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint favoring introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for nonsense-mediated decay effectors, we show that introns located at the edge of nucleosomes display higher splicing efficiency than those at the center. However, multiple regression analysis indicates that the low GC content of introns, rather than nucleosome positioning, is associated with high splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning, and intron evolution in Paramecium.