Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs (phased, small interfering RNAs) are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt (nucleotides) phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at the premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 (Dicer-like 5) for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely i) not triggered by microRNAs, ii) not loaded by AGO18 proteins, and iii) not capable of mediating PHAS precursor cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function.

Reproductive phasiRNAs are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely (i) not triggered by microRNAs, (ii) not loaded by AGO18 proteins, and (iii) not capable of mediating cis-cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known.

Plastid dsRNA transgenes trigger phased small RNA-based gene silencing of nuclear-encoded genes.

Plastid transformation technology has been widely used to express traits of potential commercial importance, though the technology has been limited to traits that function while sequestered in the organelle. Prior research indicates that plastid contents can escape from the organelle, suggesting a possible mechanism for engineering plastid transgenes to function in other cellular locations. To test this hypothesis, we created tobacco (Nicotiana tabacum cv. Petit Havana) plastid transformants that express a fragment of the nuclear-encoded Phytoene desaturase (PDS) gene capable of catalyzing post-transcriptional gene silencing if RNA escapes into the cytoplasm. We found multiple lines of direct evidence that plastid-encoded PDS transgenes affect nuclear PDS gene silencing: knockdown of the nuclear-encoded PDS mRNA and/or its apparent translational inhibition, biogenesis of 21-nucleotide (nt) phased small interfering RNAs (phasiRNAs), and pigment-deficient plants. Furthermore, plastid-expressed dsRNA with no cognate nuclear-encoded pairing partner also produced abundant 21-nt phasiRNAs in the cytoplasm, demonstrating that a nuclear-encoded template is not required for siRNA biogenesis. Our results indicate that RNA escape from plastids to the cytoplasm occurs generally, with functional consequences that include entry into the gene silencing pathway. Furthermore, we uncover a method to produce plastid-encoded traits with functions outside of the organelle and open additional fields of study in plastid development, compartmentalization, and small RNA biogenesis.

Phylogenetic analyses of seven protein families refine the evolution of small RNA pathways in green plants.

Several protein families participate in the biogenesis and function of small RNAs (sRNAs) in plants. Those with primary roles include Dicer-like (DCL), RNA-dependent RNA polymerase (RDR), and Argonaute (AGO) proteins. Protein families such as double-stranded RNA-binding (DRB), SERRATE (SE), and SUPPRESSION OF SILENCING 3 (SGS3) act as partners of DCL or RDR proteins. Here, we present curated annotations and phylogenetic analyses of seven sRNA pathway protein families performed on 196 species in the Viridiplantae (aka green plants) lineage. Our results suggest that the RDR3 proteins emerged earlier than RDR1/2/6. RDR6 is found in filamentous green algae and all land plants, suggesting that the evolution of RDR6 proteins coincides with the evolution of phased small interfering RNAs (siRNAs). We traced the origin of the 24-nt reproductive phased siRNA-associated DCL5 protein back to the American sweet flag (Acorus americanus), the earliest diverged, extant monocot species. Our analyses of AGOs identified multiple duplication events of AGO genes that were lost, retained, or further duplicated in subgroups, indicating that the evolution of AGOs is complex in monocots. The results also refine the evolution of several clades of AGO proteins, such as AGO4, AGO6, AGO17, and AGO18. Analyses of nuclear localization signal sequences and catalytic triads of AGO proteins shed light on the regulatory roles of diverse AGOs. Collectively, this work generates a curated and evolutionarily coherent annotation for gene families involved in plant sRNA biogenesis/function and provides insights into the evolution of major sRNA pathways.

A versatile enhanced freeze-substitution protocol for volume electron microscopy.

Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a routine tool for understanding fundamental and applied biological questions. One of the enabling factors for its adoption has been the development of conventional fixation protocols with improved heavy metal staining. However, freeze-substitution with organic solvent-based fixation and staining has not realized the same level of benefit. Here, we report a straightforward approach including osmium tetroxide, acetone and up to 3% water substitution fluid (compatible with traditional or fast freeze-substitution protocols), warm-up and transition from organic solvent to aqueous 2% osmium tetroxide. Once fully hydrated, samples were processed in aqueous based potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide, uranyl acetate and lead acetate before resin infiltration and polymerization. We observed a consistent and substantial increase in heavy metal staining across diverse and difficult-to-fix test organisms and tissue types, including plant tissues (Hordeum vulgare), nematode (Caenorhabditis elegans) and yeast (Saccharomyces cerevisiae). Our approach opens new possibilities to combine the benefits of cryo-preservation with enhanced contrast for volume electron microscopy in diverse organisms.

The evolutionary history of small RNAs in Solanaceae.

The Solanaceae or "nightshade" family is an economically important group with remarkable diversity. To gain a better understanding of how the unique biology of the Solanaceae relates to the family's small RNA (sRNA) genomic landscape, we downloaded over 255 publicly available sRNA data sets that comprise over 2.6 billion reads of sequence data. We applied a suite of computational tools to predict and annotate two major sRNA classes: (1) microRNAs (miRNAs), typically 20- to 22-nucleotide (nt) RNAs generated from a hairpin precursor and functioning in gene silencing and (2) short interfering RNAs (siRNAs), including 24-nt heterochromatic siRNAs typically functioning to repress repetitive regions of the genome via RNA-directed DNA methylation, as well as secondary phased siRNAs and trans-acting siRNAs generated via miRNA-directed cleavage of a polymerase II-derived RNA precursor. Our analyses described thousands of sRNA loci, including poorly understood clusters of 22-nt siRNAs that accumulate during viral infection. The birth, death, expansion, and contraction of these sRNA loci are dynamic evolutionary processes that characterize the Solanaceae family. These analyses indicate that individuals within the same genus share similar sRNA landscapes, whereas comparisons between distinct genera within the Solanaceae reveal relatively few commonalities.

The Streptochaeta Genome and the Evolution of the Grasses.

In this work, we sequenced and annotated the genome of Streptochaeta angustifolia, one of two genera in the grass subfamily Anomochlooideae, a lineage sister to all other grasses. The final assembly size is over 99% of the estimated genome size. We find good collinearity with the rice genome and have captured most of the gene space. Streptochaeta is similar to other grasses in the structure of its fruit (a caryopsis or grain) but has peculiar flowers and inflorescences that are distinct from those in the outgroups and in other grasses. To provide tools for investigations of floral structure, we analyzed two large families of transcription factors, AP2-like and R2R3 MYBs, that are known to control floral and spikelet development in rice and maize among other grasses. Many of these are also regulated by small RNAs. Structure of the gene trees showed that the well documented whole genome duplication at the origin of the grasses (ρ) occurred before the divergence of the Anomochlooideae lineage from the lineage leading to the rest of the grasses (the spikelet clade) and thus that the common ancestor of all grasses probably had two copies of the developmental genes. However, Streptochaeta (and by inference other members of Anomochlooideae) has lost one copy of many genes. The peculiar floral morphology of Streptochaeta may thus have derived from an ancestral plant that was morphologically similar to the spikelet-bearing grasses. We further identify 114 loci producing microRNAs and 89 loci generating phased, secondary siRNAs, classes of small RNAs known to be influential in transcriptional and post-transcriptional regulation of several plant functions.

GWAS identifies an ortholog of the rice D11 gene as a candidate gene for grain size in an international collection of hexaploid wheat.

Grain size is a key agronomic trait that contributes to grain yield in hexaploid wheat. Grain length and width were evaluated in an international collection of 157 wheat accessions. These accessions were genetically characterized using a genotyping-by-sequencing (GBS) protocol that produced 73,784 single nucleotide polymorphism (SNP) markers. GBS-derived genotype calls obtained on Chinese Spring proved extremely accurate when compared to the reference (> 99.9%) and showed > 95% agreement with calls made at SNP loci shared with the 90 K SNP array on a subset of 71 Canadian wheat accessions for which both types of data were available. This indicates that GBS can yield a large amount of highly accurate SNP data in hexaploid wheat. The genetic diversity analysis performed using this set of SNP markers revealed the presence of six distinct groups within this collection. A GWAS was conducted to uncover genomic regions controlling variation for grain length and width. In total, seven SNPs were found to be associated with one or both traits, identifying three quantitative trait loci (QTLs) located on chromosomes 1D, 2D and 4A. In the vicinity of the peak SNP on chromosome 2D, we found a promising candidate gene (TraesCS2D01G331100), whose rice ortholog (D11) had previously been reported to be involved in the regulation of grain size. These markers will be useful in breeding for enhanced wheat productivity.

Pre-meiotic 21-nucleotide reproductive phasiRNAs emerged in seed plants and diversified in flowering plants.

Plant small RNAs are important regulatory elements that fine-tune gene expression and maintain genome integrity by silencing transposons. Reproductive organs of monocots produce abundant phased, small interfering RNAs (phasiRNAs). The 21-nt reproductive phasiRNAs triggered by miR2118 are highly enriched in pre-meiotic anthers, and have been found in multiple eudicot species, in contrast with prior reports of monocot specificity. The 24-nt reproductive phasiRNAs are triggered by miR2275, and are highly enriched during meiosis in many angiosperms. Here, we report the widespread presence of the 21-nt reproductive phasiRNA pathway in eudicots including canonical and non-canonical microRNA (miRNA) triggers of this pathway. In eudicots, these 21-nt phasiRNAs are enriched in pre-meiotic stages, a spatiotemporal distribution consistent with that of monocots and suggesting a role in anther development. Although this pathway is apparently absent in well-studied eudicot families including the Brassicaceae, Solanaceae and Fabaceae, our work in eudicots supports an earlier singular finding in spruce, a gymnosperm, indicating that the pathway of 21-nt reproductive phasiRNAs emerged in seed plants and was lost in some lineages.

Corilagin and 1,3,6-Tri-O-galloy-ß-D-glucose: potential inhibitors of SARS-CoV-2 variants.

The COVID-19 disease caused by the virus SARS-CoV-2, first detected in December 2019, is still emerging through virus mutations. Although almost under control in some countries due to effective vaccines that are mitigating the worldwide pandemic, the urgency to develop additional vaccines and therapeutic treatments is imperative. In this work, the natural polyphenols corilagin and 1,3,6-tri-O-galloy-ß-d-glucose (TGG) are investigated to determine the structural basis of inhibitor interactions as potential candidates to inhibit SARS-CoV-2 viral entry into target cells. First, the therapeutic potential of the ligands are assessed on the ACE2/wild-type RBD. We first use molecular docking followed by molecular dynamics, to take into account the conformational flexibility that plays a significant role in ligand binding and that cannot be captured using only docking, and then analyze more precisely the affinity of these ligands using MMPBSA binding free energy. We show that both ligands bind to the ACE2/wild-type RBD interface with good affinities which might prevent the ACE2/RBD association. Second, we confirm the potency of these ligands to block the ACE2/RBD association using a combination of surface plasmon resonance and biochemical inhibition assays. These experiments confirm that TGG and, to a lesser extent, corilagin, inhibit the binding of RBD to ACE2. Both experiments and simulations show that the ligands interact preferentially with RBD, while weak binding is observed with ACE2, hence, avoiding potential physiological side-effects induced by the inhibition of ACE2. In addition to the wild-type RBD, we also study numerically three RBD mutations (E484K, N501Y and E484K/N501Y) found in the main SARS-CoV-2 variants of concerns. We find that corilagin could be as effective for RBD/E484K but less effective for the RBD/N501Y and RBD/E484K-N501Y mutants, while TGG strongly binds at relevant locations to all three mutants, demonstrating the significant interest of these molecules as potential inhibitors for variants of SARS-CoV-2.

Premeiotic, 24-Nucleotide Reproductive PhasiRNAs Are Abundant in Anthers of Wheat and Barley But Not Rice and Maize.

Two classes of premeiotic (21-nucleotides [nt]) and meiotic (24-nt) phased small interfering RNAs (phasiRNAs) and their patterns of accumulation have been described in maize (Zea mays) and rice (Oryza sativa) anthers. Their precise function remains unclear, but studies have shown that they support male fertility. The important role of phasiRNAs in anthers underpins our present study to characterize these small RNAs in wheat (Triticum aestivum) and barley (Hordeum vulgare) anthers. We staged anthers at every 0.2 mm of development for one wheat and two barley varieties. We isolated premeiotic (0.2, 0.4, and 0.6 mm), meiotic (0.8, 1.0, and 1.4 mm), and postmeiotic (1.8 mm) anthers, for which we then investigated accumulation patterns of RNAs, including reproductive phasiRNAs. We annotated a total of 12,821 and 2,897 PHAS loci in the wheat and barley genomes, respectively. By comparing the total number of PHAS loci in genomes of maize, rice, barley, and wheat, we identified an expansion of reproductive PHAS loci in the genomes of Poaceae subfamilies from Panicoideae to Oryzoideae and to Poideae. In addition to the two classes of premeiotic (21-nt) and meiotic (24-nt) phasiRNAs, previously described in maize and rice anthers, we characterized a group of 24-nt phasiRNAs that accumulate in premeiotic anthers. The absence of premeiotic 24-nt phasiRNAs in maize and rice suggests a divergence in grass species of the Poideae subfamily. Additionally, we performed a gene coexpression analysis describing the regulation of phasiRNA biogenesis in wheat and barley anthers. We highlight Argonaute 9 (AGO9) and Argonaute 6 (AGO6) as candidate binding partners of premeiotic and meiotic 24-nt phasiRNAs, respectively.

The commitment of barley microspores into embryogenesis correlates with miRNA-directed regulation of members of the SPL, GRF and HD-ZIPIII transcription factor families.

Microspore embryogenesis is a model for developmental plasticity and cell fate decisions. To investigate the role of miRNAs in this development, we sequenced sRNAs and the degradome of barley microspores collected prior to (day 0) and after (days 2 and 5) the application of a stress treatment known to induce embryogenesis. Microspores isolated at these timepoints were uniform in both appearance and in their complements of sRNAs. We detected 68 miRNAs in microspores. The abundance of 51 of these miRNAs differed significantly during microspore development. One group of miRNAs was induced when the stress treatment was applied, prior to being repressed when microspores transitioned to embryogenesis. Another group of miRNAs were up-regulated in day-2 microspores and their abundance remained stable or increased in day-5 microspores, a timepoint at which the first clear indications of the transition toward embryogenesis were visible. Collectively, these miRNAs might play a role in the modulation of the stress response, the repression of gametic development, and/or the gain of embryogenic potential. A degradome analysis allowed us to validate the role of miRNAs in regulating 41 specific transcripts. We showed that the transition of microspores toward the embryogenesis pathway involves miRNA-directed regulation of members of the ARF, SPL, GRF, and HD-ZIPIII transcription factor families. We noted that 41.5% of these targets were shared between day-2 and day-5 microspores while 26.8% were unique to day-5 microspores. The former set may act to disrupt transcripts involved in pollen development while the latter set may drive the commitment to embryogenesis.

QTL mapping uncovers a semi-dwarf 1 (sdw1) allele in the barley (Hordeum vulgare) ND23049 line.

In barley, semi-dwarf varieties are attractive for their superior harvest index and lodging resistance, but many semi-dwarf barley genotypes suffer from poor spike emergence. We performed a genetic characterization of a semi-dwarf line (ND23049) that combines short stature, strong stiff culms, and adequate spike emergence. We developed a doubled haploid (DH) population derived by crossing ND23049 and the cultivar CLE253. A subset of 88 DH lines and parents were characterized for plant height in 2013 and 2014 and genotyped. In total, 1984 SNPs (345 unique loci) were used to produce a linkage map of 1127.1 cM. Three QTLs for plant height were detected in this population and coincided with the HvGA20ox2/Sdw1, HvBRI1/Uzu1, and HvPRR95 gene loci. The phenotypic variation explained by each QTL was 75.8%, 7.7%, and 4.1%, respectively, and jointly explained 83.3% (2013) and 87.7% (2014) of plant height. Our results suggest that ND23049 contributed the "short" allele at the HvGA20ox2/sdw1 locus while CLE253 provided "short" alleles at the HvBRI1/uzu1 and HvPRR95 loci. We identified a large deletion (at least 92.7 Kb), including HvGA20ox2 (Sdw1), as the causal mutation in ND23049. A set of tightly flanked SNP markers will help breeders to develop improved semi-dwarf varieties.

Differential Expression Profiling of Microspores During the Early Stages of Isolated Microspore Culture Using the Responsive Barley Cultivar Gobernadora.

In barley, it is possible to induce embryogenesis in the haploid and uninucleate microspore to obtain a diploid plant that is perfectly homozygous. To change developmental fates in this fashion, microspores need to engage in cellular de-differentiation, interrupting the pollen formation, and restore totipotency prior to engaging in embryogenesis. In this work, we used the barley cultivar Gobernadora to characterize the transcriptome of microspores prior to (day 0) and immediately after (days 2 and 5) the application of a stress pretreatment. A deep RNA-seq analysis revealed that microspores at these three time points exhibit a transcriptome of ∼14k genes, ∼90% of which were shared. An expression analysis identified a total of 3,382 differentially expressed genes (DEGs); of these, 2,155 and 2,281 DEGs were respectively identified when contrasting expression at days 0 and 2 and at days 2 and 5. These define 8 expression profiles in which DEGs share a common up- or down-regulation at these time points. Up-regulation of numerous glutathione S-transferase and heat shock protein genes as well as down-regulation of ribosomal subunit protein genes was observed between days 0 and 2. The transition from microspores to developing embryos (days 2 vs. 5) was marked by the induction of transcription factor genes known to play important roles in early embryogenesis, numerous genes involved in hormone biosynthesis and plant hormonal signal transduction in addition to genes involved in secondary metabolism. This work sheds light on transcriptional changes accompanying an important developmental shift and provides candidate biomarkers for embryogenesis in barley.

Complete Genome Sequences of 12 Isolates of Listeria monocytogenes Belonging to Serotypes 1/2a, 1/2b, and 4b Obtained from Food Products and Food-Processing Environments in Canada.

Listeria monocytogenes is the etiological agent for an often fatal foodborne illness known as listeriosis. Here, we present the complete genome sequences of 12 L. monocytogenes isolates representing the three most common serotypes of this pathogen (1/2a, 1/2b, and 4b), collected in Canada from different food products and environmental sources.

Genotyping-by-Sequencing on Pooled Samples and its Use in Measuring Segregation Bias during the Course of Androgenesis in Barley.

Estimation of allelic frequencies is often required in breeding but genotyping many individuals at many loci can be expensive. We have developed a genotyping-by-sequencing (GBS) approach for estimating allelic frequencies on pooled samples (Pool-GBS) and used it to examine segregation distortion in doubled haploid (DH) populations of barley ( L.). In the first phase, we genotyped each line individually and exploited these data to explore a strategy to call single nucleotide polymorphisms (SNPs) on pooled reads. We measured both the number of SNPs called and the variance of the estimated allelic frequencies at various depths of coverage on a subset of reads containing 5 to 25 million reads. We show that allelic frequencies could be cost-effectively and accurately estimated at a depth of 50 reads per SNP using 15 million reads. This Pool-GBS approach yielded 1984 SNPs whose allelic frequency estimates were highly reproducible (CV = 10.4%) and correlated ( = 0.9167) with the "true" frequency derived from analysis of individual lines. In a second phase, we used Pool-GBS to investigate segregation bias throughout androgenesis from microspores to a population of regenerated plants. No strong bias was detected among the microspores resulting from the meiotic divisions, whereas significant biases could be shown to arise during embryo formation and plant regeneration. In summary, this methodology provides an approach to estimate allelic frequencies more efficiently and on materials that are unsuitable for individual analysis. In addition, it allowed us to shed light on the process of androgenesis in barley.

Extent and overlap of segregation distortion regions in 12 barley crosses determined via a Pool-GBS approach.

KEY MESSAGE: Extent and overlap of segregation distortion regions in 12 barley crosses determined via a Pool-GBS approach. Segregation distortion is undesirable as it alters the frequency of alleles and can reduce the chances of obtaining a particular combination of alleles. In this work, we have used a pooled genotyping-by-sequencing (Pool-GBS) approach to estimate allelic frequencies and used it to examine segregation distortion in 12 segregating populations of barley derived from androgenesis. Thanks to the extensive genome-wide SNP coverage achieved (between 674 and 1744 markers), we determined that the proportion of distorted markers averaged 28.9 % while 25.3 % of the genetic map fell within segregation distortion regions (SDRs). These SDRs were characterized and identified based on the position of the marker showing the largest distortion and the span of each SDR. Summed across all 12 crosses, 36 different SDR peaks could be distinguished from a total of 50 SDRs and a majority of these SDRs (27 of 36) were observed in only one population. While most shared SDRs were common to only two crosses, two SDRs (SDR3.1 and SDR4.2) were exceptionally recurrent (seen in five and four crosses, respectively). Because of the broad span of most SDRs, an average of 30 % of crosses showed segregation distortion in any given chromosomal segment. In reciprocal crosses, although some SDRs were clearly shared, others were unique to a single direction. In summary, segregation distortion is highly variable in its extent and the number of loci underpinning these distortions seems to be quite large even in a narrow germplasm such as six-row spring barley.

Development of a new molecular subtyping tool for Salmonella enterica serovar Enteritidis based on single nucleotide polymorphism genotyping using PCR.

The lack of a sufficiently discriminatory molecular subtyping tool for Salmonella enterica serovar Enteritidis has hindered source attribution efforts and impeded regulatory actions required to disrupt its food-borne transmission. The underlying biological reason for the ineffectiveness of current molecular subtyping tools such as pulsed-field gel electrophoresis (PFGE) and phage typing appears to be related to the high degree of clonality of S. Enteritidis. By interrogating the organism's genome, we previously identified single nucleotide polymorphisms (SNP) distributed throughout the chromosome and have designed a highly discriminatory PCR-based SNP typing test based on 60 polymorphic loci. The application of the SNP-PCR method to DNA samples from S. Enteritidis strains (n = 55) obtained from a variety of sources has led to the differentiation and clustering of the S. Enteritidis isolates into 12 clades made up of 2 to 9 isolates per clade. Significantly, the SNP-PCR assay was able to further differentiate predominant PFGE types (e.g., XAI.0003) and phage types (e.g., phage type 8) into smaller subsets. The SNP-PCR subtyping test proved to be an accurate, precise, and quantitative tool for evaluating the relationships among the S. Enteritidis isolates tested in this study and should prove useful for clustering related S. Enteritidis isolates involved in outbreaks.

High resolution assembly and characterization of genomes of Canadian isolates of Salmonella Enteritidis.

BACKGROUND: There is a need to characterize genomes of the foodborne pathogen, Salmonella enterica serovar Enteritidis (SE) and identify genetic information that could be ultimately deployed for differentiating strains of the organism, a need that is yet to be addressed mainly because of the high degree of clonality of the organism. In an effort to achieve the first characterization of the genomes of SE of Canadian origin, we carried out massively parallel sequencing of the nucleotide sequence of 11 SE isolates obtained from poultry production environments (n = 9), a clam and a chicken, assembled finished genomes and investigated diversity of the SE genome. RESULTS: The median genome size was 4,678,683 bp. A total of 4,833 chromosomal genes defined the pan genome of our field SE isolates consisting of 4,600 genes present in all the genomes, i.e., core genome, and 233 genes absent in at least one genome (accessory genome). Genome diversity was demonstrable by the presence of 1,360 loci showing single nucleotide polymorphism (SNP) in the core genome which was used to portray the genetic distances by means of a phylogenetic tree for the SE isolates. The accessory genome consisted mostly of previously identified SE prophage sequences as well as two, apparently full-sized, novel prophages namely a 28 kb sequence provisionally designated as SE-OLF-10058 (3) prophage and a 43 kb sequence provisionally designated as SE-OLF-10012 prophage. CONCLUSIONS: The number of SNPs identified in the relatively large core genome of SE is a reflection of substantial diversity that could be exploited for strain differentiation as shown by the development of an informative phylogenetic tree. Prophage sequences can also be exploited for SE strain differentiation and lineage tracking. This work has laid the ground work for further studies to develop a readily adoptable laboratory test for the subtyping of SE.

Effect of temperature and tree species on damage progression caused by whitespotted sawyer (Coleoptera: Cerambycidae) larvae in recently burned logs.

The whitespotted sawyer, Monochamus scutellatus scutellatus (Say) (Coleoptera: Ce-rambycidae), is one of the most damaging wood-boring insects in recently burned boreal forests of North America. In Canada, salvage logging after wildfire contributes to maintaining the timber volume required by the forest industry, but larvae of this insect cause significant damage that reduces the economic value of lumber products. This study aimed to estimate damage progression as a function of temperature in recently burned black spruce (Picea mariana (Miller) Britton, Sterns, and Poggenburg) and jack pine (Pinus banksiana Lambert) trees. Using axial tomographic technology, we modeled subcortical development and gallery depth progression rates as functions of temperature for both tree species. Generally, these rates were slightly faster in black spruce than in jack pine logs. Eggs laid on logs kept at 12 degrees C did not hatch or larvae were unable to establish themselves under the bark because no larval development was observed. At 16 degrees C, larvae stayed under the bark for > 200 d before penetrating into the sapwood. At 20 degrees C, half of the larvae entered the sapwood after 30-50 d, but gallery depth progression stopped for approximately 70 d, suggesting that larvae went into diapause. The other half of the larvae entered the sapwood only after 100-200 d. At 24 and 28 degrees C, larvae entered the sapwood after 26-27 and 21 d, respectively. At 28 degrees C, gallery depth progressed at a rate of 1.44 mm/d. Temperature threshold for subcortical development was slightly lower in black spruce (12.9 degrees C) than in jack pine (14.6 degrees C) and it was 1 degrees C warmer for gallery depth progression for both tree species. These results indicate that significant damage may occur within a few months after fire during warm summers, particularly in black spruce, which highlights the importance of beginning postfire salvage logging as soon as possible to reduce economic losses.