Investigating Grapevine Red Blotch Virus Infection in Vitis vinifera L. cv. Cabernet Sauvignon Grapes: A Multi-Omics Approach.

Grapevine red blotch virus (GRBV) is a recently identified virus. Previous research indicates primarily a substantial impact on berry ripening in all varieties studied. The current study analyzed grapes' primary and secondary metabolism across grapevine genotypes and seasons to reveal both conserved and variable impacts to GRBV infection. Vitis vinifera cv. Cabernet Sauvignon (CS) grapevines grafted on two different rootstocks (110R and 420A) were analyzed in 2016 and 2017. Metabolite profiling revealed a considerable impact on amino acid and malate acid levels, volatile aroma compounds derived from the lipoxygenase pathway, and anthocyanins synthesized in the phenylpropanoid pathway. Conserved transcriptional responses to GRBV showed induction of auxin-mediated pathways and photosynthesis with inhibition of transcription and translation processes mainly at harvest. There was an induction of plant-pathogen interactions at pre-veraison, for all genotypes and seasons, except for CS 110R in 2017. Lastly, differential co-expression analysis revealed a transcriptional shift from metabolic synthesis and energy metabolism to transcription and translation processes associated with a virus-induced gene silencing transcript. This plant-derived defense response transcript was only significantly upregulated at veraison for all genotypes and seasons, suggesting a phenological association with disease expression and plant immune responses.

The walnut (Juglans regia) genome sequence reveals diversity in genes coding for the biosynthesis of non-structural polyphenols.

The Persian walnut (Juglans regia L.), a diploid species native to the mountainous regions of Central Asia, is the major walnut species cultivated for nut production and is one of the most widespread tree nut species in the world. The high nutritional value of J. regia nuts is associated with a rich array of polyphenolic compounds, whose complete biosynthetic pathways are still unknown. A J. regia genome sequence was obtained from the cultivar 'Chandler' to discover target genes and additional unknown genes. The 667-Mbp genome was assembled using two different methods (SOAPdenovo2 and MaSuRCA), with an N50 scaffold size of 464 955 bp (based on a genome size of 606 Mbp), 221 640 contigs and a GC content of 37%. Annotation with MAKER-P and other genomic resources yielded 32 498 gene models. Previous studies in walnut relying on tissue-specific methods have only identified a single polyphenol oxidase (PPO) gene (JrPPO1). Enabled by the J. regia genome sequence, a second homolog of PPO (JrPPO2) was discovered. In addition, about 130 genes in the large gallate 1-ß-glucosyltransferase (GGT) superfamily were detected. Specifically, two genes, JrGGT1 and JrGGT2, were significantly homologous to the GGT from Quercus robur (QrGGT), which is involved in the synthesis of 1-O-galloyl-ß-d-glucose, a precursor for the synthesis of hydrolysable tannins. The reference genome for J. regia provides meaningful insight into the complex pathways required for the synthesis of polyphenols. The walnut genome sequence provides important tools and methods to accelerate breeding and to facilitate the genetic dissection of complex traits.

Effects of high temperatures on threatened estuarine fishes during periods of extreme drought.

Climate change and associated increases in water temperatures may impact physiological performance in ectotherms and exacerbate endangered species declines. We used an integrative approach to assess the impact of elevated water temperature on two fishes of immediate conservation concern in a large estuary system, the threatened longfin smelt (Spirinchus thaleichthys) and endangered delta smelt (Hypomesus transpacificus). Abundances have reached record lows in California, USA, and these populations are at imminent risk of extirpation. California is currently impacted by a severe drought, resulting in high water temperatures, conditions that will become more common as a result of climate change. We exposed fish to environmentally relevant temperatures (14°C and 20°C) and used RNA sequencing to examine the transcriptome-wide responses to elevated water temperature in both species. Consistent with having a lower temperature tolerance, longfin smelt exhibited a pronounced cellular stress response, with an upregulation of heat shock proteins, after exposure to 20°C that was not observed in delta smelt. We detected an increase in metabolic rate in delta smelt at 20°C and increased expression of genes involved in metabolic processes and protein synthesis, patterns not observed in longfin smelt. Through examination of responses across multiple levels of biological organization, and by linking these responses to habitat distributions in the wild, we demonstrate that longfin smelt may be more susceptible than delta smelt to increases in temperatures, and they have little room to tolerate future warming in California. Understanding the species-specific physiological responses of sensitive species to environmental stressors is crucial for conservation efforts and managing aquatic systems globally.

Stacking resistance to crown gall and nematodes in walnut rootstocks.

BACKGROUND: Crown gall (CG) (Agrobacterium tumefaciens) and the root lesion nematodes (RLNs) (Pratylenchus vulnus) are major challenges faced by the California walnut industry, reducing productivity and increasing the cost of establishing and maintaining orchards. Current nematode control strategies include nematicides, crop rotation, and tolerant cultivars, but these methods have limits. Developing genetic resistance through novel approaches like RNA interference (RNAi) can address these problems. RNAi-mediated silencing of CG disease in walnut (Juglans regia L.) has been achieved previously. We sought to place both CG and nematode resistance into a single walnut rootstock genotype using co-transformation to stack the resistance genes. A. tumefaciens, carrying self-complimentary iaaM and ipt transgenes, and Agrobacterium rhizogenes, carrying a self-complimentary Pv010 gene from P. vulnus, were used as co-transformation vectors. RolABC genes were introduced by the resident T-DNA in the A. rhizogenes Ri-plasmid used as a vector for plant transformation. Pv010 and Pv194 (transgenic control) genes were also transferred separately using A. tumefaciens. To test for resistance, transformed walnut roots were challenged with P. vulnus and microshoots were challenged with a virulent strain of A. tumefaciens. RESULTS: Combining the two bacterial strains at a 1:1 rather than 1:3 ratio increased the co-transformation efficiency. Although complete immunity to nematode infection was not observed, transgenic lines yielded up to 79% fewer nematodes per root following in vitro co-culture than untransformed controls. Transgenic line 33-3-1 exhibited complete crown gall control and 32% fewer nematodes. The transgenic plants had thicker, longer roots than untransformed controls possibly due to insertion of rolABC genes. When the Pv010 gene was present in roots with or without rolABC genes there was partial or complete control of RLNs. Transformation using only one vector showed 100% control in some lines. CONCLUSIONS: CG and nematode resistance gene stacking controlled CG and RLNs simultaneously in walnuts. Silencing genes encoding iaaM, ipt, and Pv010 decrease CG formation and RLNs populations in walnut. Beneficial plant genotype and phenotype changes are caused by co-transformation using A. tumefaciens and A. rhizogenes strains. Viable resistance against root lesion nematodes in walnut plants may be accomplished in the future using this gene stacking technology.

Probing functional polymorphisms in the dengue vector, Aedes aegypti.

BACKGROUND: Dengue is the most prevalent arboviral disease world-wide and its primary vector is the mosquito Aedes aegypti. The current lack of commercially-available vaccines makes control of vector populations the only effective strategy to prevent dengue transmission. Aedes aegypti geographic populations exhibit great variability in insecticide resistance and susceptibility to dengue infection. The characterization of single nucleotide polymorphisms (SNPs) as molecular markers to study quantitatively this variation is needed greatly because this species has a low abundance of microsatellite markers and limited known restriction fragments length polymorphisms (RFLPs) and single-strand conformation polymorphism (SSCP) markers. RESULTS: We used RNA-seq to characterize SNPs in three Ae. aegypti strains, including the Liverpool (LVP) strain, from which the current genome annotation is derived. We identified 131,764 unique genome locations with at least one alternative nucleotide to what is reported in the reference annotation. These comprised changes in both open-reading frames (ORFs) and untranslated regions (UTRs) of transcripts. An in depth-look at sequence variation in immunity genes revealed that those associated with autophagy, MD2-like receptors and Peptidoglycan Recognition Proteins had more sequence variation in their 3'UTRs than mutations associated with non-synonymous changes. This supports the conclusion that these genes had maintained their functional specificity while being adapted to different regulatory domains. In contrast, a number of peroxidases, serpins and Clip-domain serine proteases exhibited conservation of putative UTR regulatory sequences while displaying diversification of the ORFs. Transcriptome evidence also was found for ~2500 novel transcriptional units (NTUs) not annotated in the reference genome. CONCLUSIONS: The transcriptome-wide assessment of within and inter-strain polymorphisms in Ae. aegypti adds considerably to the number of molecular markers available for genetic studies in this mosquito. Additionally, data supporting NTU discovery emphasizes the need for continuous amendments of the reference genome annotation.

Genome-wide SNP discovery in walnut with an AGSNP pipeline updated for SNP discovery in allogamous organisms.

BACKGROUND: A genome-wide set of single nucleotide polymorphisms (SNPs) is a valuable resource in genetic research and breeding and is usually developed by re-sequencing a genome. If a genome sequence is not available, an alternative strategy must be used. We previously reported the development of a pipeline (AGSNP) for genome-wide SNP discovery in coding sequences and other single-copy DNA without a complete genome sequence in self-pollinating (autogamous) plants. Here we updated this pipeline for SNP discovery in outcrossing (allogamous) species and demonstrated its efficacy in SNP discovery in walnut (Juglans regia L.). RESULTS: The first step in the original implementation of the AGSNP pipeline was the construction of a reference sequence and the identification of single-copy sequences in it. To identify single-copy sequences, multiple genome equivalents of short SOLiD reads of another individual were mapped to shallow genome coverage of long Sanger or Roche 454 reads making up the reference sequence. The relative depth of SOLiD reads was used to filter out repeated sequences from single-copy sequences in the reference sequence. The second step was a search for SNPs between SOLiD reads and the reference sequence. Polymorphism within the mapped SOLiD reads would have precluded SNP discovery; hence both individuals had to be homozygous. The AGSNP pipeline was updated here for using SOLiD or other type of short reads of a heterozygous individual for these two principal steps. A total of 32.6X walnut genome equivalents of SOLiD reads of vegetatively propagated walnut scion cultivar 'Chandler' were mapped to 48,661 'Chandler' bacterial artificial chromosome (BAC) end sequences (BESs) produced by Sanger sequencing during the construction of a walnut physical map. A total of 22,799 putative SNPs were initially identified. A total of 6,000 Infinium II type SNPs evenly distributed along the walnut physical map were selected for the construction of an Infinium BeadChip, which was used to genotype a walnut mapping population having 'Chandler' as one of the parents. Genotyping results were used to adjust the filtering parameters of the updated AGSNP pipeline. With the adjusted filtering criteria, 69.6% of SNPs discovered with the updated pipeline were real and could be mapped on the walnut genetic map. A total of 13,439 SNPs were discovered by BES re-sequencing. BESs harboring SNPs were in 677 FPC contigs covering 98% of the physical map of the walnut genome. CONCLUSION: The updated AGSNP pipeline is a versatile SNP discovery tool for a high-throughput, genome-wide SNP discovery in both autogamous and allogamous species. With this pipeline, a large set of SNPs were identified in a single walnut cultivar.

Transcriptomic changes underlying glucocorticoid-induced suppression of milk production by dairy cows.

Milk production by dairy cows is sensitive to increased levels of stress hormones such as glucocorticoids (GC) that also regulate the transcription of several genes required for milk synthesis. Whereas previous studies identified that an exogenous GC such as dexamethasone (DEX) transiently suppresses milk yield in several species without any pronounced effect on milk protein or fat percentage, the mechanism underlying this effect has not been established. In this study we sought to establish changes within the mammary glands of non-pregnant dairy cows in their second lactation (n = 3-4; 648-838 kg) following a single dose of exogenous DEX. Changes in the udder were monitored by serial biopsy of alternating quarters, concurrent with quarter-level monitoring of milk yield and composition. Dexamethasone increased serum glucose levels from 12-36 h (p <0 .05), reduced milk yield from 12-48 h (p <0 .05), increased % milk protein content at 24 h post-DEX, and transiently decreased both milk lactose and α-lactalbumin content, while not altering the level of milk fat. After 72 h, all aspects of milk production had returned to pre-treatment levels. Transcriptomic changes in the mammary glands in response to DEX were identified by RNA sequencing followed by differential gene expression analysis. Coincident with the milk yield and composition changes was the differential expression of 519 and 320 genes at 12 and 24 h after DEX (adjusted p <0 .05), respectively, with the return of all gene expression to baseline levels by 72 h. Among the transcriptomic changes in response to DEX, there was notable downregulation of elements in the lactose synthesis pathway, specifically AQP3, GALE and LALBA (α-lactalbumin) at 12 h, and sustained downregulation of LALBA at 24 h. One gene in the pathway, UGP2, was upregulated at 12-24 h post-DEX. This work supports the hypothesis that there is a direct relationship between the response to DEX and the concurrent suppression of milk yield due to the reduced synthesis of α-lactalbumin and lactose by the mammary epithelium. The ability of glucocorticoids to modulate the homeorrhetic requirements for glucose during stressful states concurrent with immune activation bears significance for dairy animals as well as a broad range of lactating mammals.

Comparative Transcriptional Analyses in the Nucleus Accumbens Identifies RGS2 as a Key Mediator of Depression-Related Behavior.

BACKGROUND: Major depressive disorder is one of the most commonly diagnosed mental illnesses worldwide, with a higher prevalence in women than in men. Although currently available pharmacological therapeutics help many individuals, they are not effective for most. Animal models have been important for the discovery of molecular alterations in stress and depression, but difficulties in adapting animal models of depression for females has impeded progress in developing novel therapeutic treatments that may be more efficacious for women. METHODS: Using the California mouse social defeat model, we took a multidisciplinary approach to identify stress-sensitive molecular targets that have translational relevance for women. We determined the impact of stress on transcriptional profiles in male and female California mouse nucleus accumbens (NAc) and compared these results with data from postmortem samples of the NAc from men and women diagnosed with major depressive disorder. RESULTS: Our cross-species computational analyses identified Rgs2 (regulator of G protein signaling 2) as a transcript downregulated by social defeat stress in female California mice and in women with major depressive disorder. RGS2 plays a key role in signal regulation of neuropeptide and neurotransmitter receptors. Viral vector-mediated overexpression of Rgs2 in the NAc restored social approach and sucrose preference in stressed female California mice. CONCLUSIONS: These studies show that Rgs2 acting in the NAc has functional properties that translate to changes in anxiety- and depression-related behavior. Future studies should investigate whether targeting Rgs2 represents a novel target for treatment-resistant depression in women.

Exploring the Transcriptome Landscape of Pomegranate Fruit Peel for Natural Product Biosynthetic Gene and SSR Marker Discovery(F).

Pomegranate fruit peel is rich in bioactive plant natural products, such as hydrolyzable tannins and anthocyanins. Despite their documented roles in human nutrition and fruit quality, genes involved in natural product biosynthesis have not been cloned from pomegranate and very little sequence information is available on pomegranate in the public domain. Shotgun transcriptome sequencing of pomegranate fruit peel cDNA was performed using RNA-Seq on the Illumina Genome Analyzer platform. Over 100 million raw sequence reads were obtained and assembled into 9,839 transcriptome assemblies (TAs) (>200 bp). Candidate genes for hydrolyzable tannin, anthocyanin, flavonoid, terpenoid and fatty acid biosynthesis and/or regulation were identified. Three lipid transfer proteins were obtained that may contribute to the previously reported IgE reactivity of pomegranate fruit extracts. In addition, 115 SSR markers were identified from the pomegranate fruit peel transcriptome and primers were designed for 77 SSR markers. The pomegranate fruit peel transcriptome set provides a valuable platform for natural product biosynthetic gene and SSR marker discovery in pomegranate. This work also demonstrates that next-generation transcriptome sequencing is an economical and effective approach for investigating natural product biosynthesis, identifying genes controlling important agronomic traits, and discovering molecular markers in non-model specialty crop species.

Association of the Lactase Persistence Haplotype Block With Disease Risk in Populations of European Descent.

Among people of European descent, the ability to digest lactose into adulthood arose via strong positive selection of a highly advantageous allele encompassing the lactase gene. Lactose-tolerant and intolerant individuals may have different disease risks due to the shared genetics of their haplotype block. Therefore, the overall objective of the study was to assess the genetic association of the lactase persistence haplotype to disease risk. Using data from the 1000Genomes project, we estimated the size of the lactase persistence haplotype block to be 1.9 Mbp containing up to 9 protein-coding genes and a microRNA. Based on the function of the genes and microRNA, we studied health phenotypes likely to be impacted by the lactase persistence allele: prostate cancer status, cardiovascular disease status, and bone mineral density. We used summary statistics from large genome-wide metanalyses-32,965 bone mineral density, 140,306 prostate cancer and 184,305 coronary artery disease subjects-to evaluate whether the lactase persistence allele was associated with these disease phenotypes. Despite the fact that previous work demonstrated that the lactase persistence haplotype block harbors increased deleterious mutations, these results suggest little effect on the studied disease phenotypes.

A Reference Genome Sequence for Giant Sequoia.

The giant sequoia (Sequoiadendron giganteum) of California are massive, long-lived trees that grow along the U.S. Sierra Nevada mountains. Genomic data are limited in giant sequoia and producing a reference genome sequence has been an important goal to allow marker development for restoration and management. Using deep-coverage Illumina and Oxford Nanopore sequencing, combined with Dovetail chromosome conformation capture libraries, the genome was assembled into eleven chromosome-scale scaffolds containing 8.125 Gbp of sequence. Iso-Seq transcripts, assembled from three distinct tissues, was used as evidence to annotate a total of 41,632 protein-coding genes. The genome was found to contain, distributed unevenly across all 11 chromosomes and in 63 orthogroups, over 900 complete or partial predicted NLR genes, of which 375 are supported by annotation derived from protein evidence and gene modeling. This giant sequoia reference genome sequence represents the first genome sequenced in the Cupressaceae family, and lays a foundation for using genomic tools to aid in giant sequoia conservation and management.

Transcriptomic response to elevated water temperatures in adult migrating Yukon River Chinook salmon (Oncorhynchus tshawytscha).

Chinook salmon (Oncorhynchus tshawytscha) declines are widespread and may be attributed, at least in part, to warming river temperatures. Water temperatures in the Yukon River and tributaries often exceed 18°C, a threshold commonly associated with heat stress and elevated mortality in Pacific salmon. Untangling the complex web of direct and indirect physiological effects of heat stress on salmon is difficult in a natural setting with innumerable system challenges but is necessary to increase our understanding of both lethal and sublethal impacts of heat stress on populations. The goal of this study was to characterize the cellular stress response in multiple Chinook salmon tissues after acute elevated temperature challenges. We conducted a controlled 4-hour temperature exposure (control, 18°C and 21°C) experiment on the bank of the Yukon River followed by gene expression (GE) profiling using a 3'-Tag-RNA-Seq protocol. The full transcriptome was analysed for 22 Chinook salmon in muscle, gill and liver tissue. Both the 21°C and 18°C treatments induced greater activity in genes associated with protein folding (e.g. HSP70, HSP90 mRNA) processes in all tissues. Global GE patterns indicate that transcriptomic responses to heat stress were highly tissue-specific, underscoring the importance of analyzing multiple tissues for determination of physiological effect. Primary superclusters (i.e. groupings of loosely related terms) of altered biological processes were identified in each tissue type, including regulation of DNA damage response (gill), regulation by host of viral transcription (liver) and regulation of the force of heart contraction (muscle) in the 21°C treatment. This study provides insight into mechanisms potentially affecting adult Chinook salmon as they encounter warm water during their spawning migration in the Yukon River and suggests that both basic and more specialized cellular functions may be disrupted.

High-quality chromosome-scale assembly of the walnut (Juglans regia L.) reference genome.

BACKGROUND: The release of the first reference genome of walnut (Juglans regia L.) enabled many achievements in the characterization of walnut genetic and functional variation. However, it is highly fragmented, preventing the integration of genetic, transcriptomic, and proteomic information to fully elucidate walnut biological processes. FINDINGS: Here, we report the new chromosome-scale assembly of the walnut reference genome (Chandler v2.0) obtained by combining Oxford Nanopore long-read sequencing with chromosome conformation capture (Hi-C) technology. Relative to the previous reference genome, the new assembly features an 84.4-fold increase in N50 size, with the 16 chromosomal pseudomolecules assembled and representing 95% of its total length. Using full-length transcripts from single-molecule real-time sequencing, we predicted 37,554 gene models, with a mean gene length higher than the previous gene annotations. Most of the new protein-coding genes (90%) present both start and stop codons, which represents a significant improvement compared with Chandler v1.0 (only 48%). We then tested the potential impact of the new chromosome-level genome on different areas of walnut research. By studying the proteome changes occurring during male flower development, we observed that the virtual proteome obtained from Chandler v2.0 presents fewer artifacts than the previous reference genome, enabling the identification of a new potential pollen allergen in walnut. Also, the new chromosome-scale genome facilitates in-depth studies of intraspecies genetic diversity by revealing previously undetected autozygous regions in Chandler, likely resulting from inbreeding, and 195 genomic regions highly differentiated between Western and Eastern walnut cultivars. CONCLUSION: Overall, Chandler v2.0 will serve as a valuable resource to better understand and explore walnut biology.

Viral genome integration of canine papillomavirus 16.

Papillomaviruses infect humans and animals, most often causing benign proliferations on skin or mucosal surfaces. Rarely, these infections persist and progress to cancer. In humans, this transformation most often occurs with high-risk papillomaviruses, where viral integration is a critical event in carcinogenesis. The first aim of this study was to sequence the viral genome of canine papillomavirus (CPV) 16 from a pigmented viral plaque that progressed to metastatic squamous cell carcinoma in a dog. The second aim was to characterize multiple viral genomic deletions and translocations as well as host integration sites. The full viral genome was identified using a combination of PCR and high throughput sequencing. CPV16 is most closely related to chipapillomaviruses CPV4, CPV9, and CPV12 and we propose CPV16 be classified as a chipapillomavirus. Assembly of the full viral genome enabled identification of deletion of portions of the E1 and E2/E4 genes and two viral translocations within the squamous cell carcinoma. Genome walking was performed which identified four sites of viral integration into the host genome. This is the first description of integration of a canine papillomavirus into the host genome, raising the possibility that CPV16 may be a potential canine high-risk papillomavirus type.

Mitochondrial oxidative stress-induced transcript variants of ATF3 mediate lipotoxic brain microvascular injury.

Elevation of blood triglycerides, primarily triglyceride-rich lipoproteins (TGRL), is an independent risk factor for cardiovascular disease and vascular dementia (VaD). Accumulating evidence indicates that both atherosclerosis and VaD are linked to vascular inflammation. However, the role of TGRL in vascular inflammation, which increases risk for VaD, remains largely unknown and its underlying mechanisms are still unclear. We strived to determine the effects of postprandial TGRL exposure on brain microvascular endothelial cells, the potential risk factor of vascular inflammation, resulting in VaD. We showed in Aung et al., J Lipid Res., 2016 that postprandial TGRL lipolysis products (TL) activate mitochondrial reactive oxygen species (ROS) and increase the expression of the stress-responsive protein, activating transcription factor 3 (ATF3), which injures human brain microvascular endothelial cells (HBMECs) in vitro. In this study, we deployed high-throughput sequencing (HTS)-based RNA sequencing methods and mito stress and glycolytic rate assays with an Agilent Seahorse XF analyzer and profiled the differential expression of transcripts, constructed signaling pathways, and measured mitochondrial respiration, ATP production, proton leak, and glycolysis of HBMECs treated with TL. Conclusions: TL potentiate ROS by mitochondria which activate mitochondrial oxidative stress, decrease ATP production, increase mitochondrial proton leak and glycolysis rate, and mitochondria DNA damage. Additionally, CPT1A1 siRNA knockdown suppresses oxidative stress and prevents mitochondrial dysfunction and vascular inflammation in TL treated HBMECs. TL activates ATF3-MAPKinase, TNF, and NRF2 signaling pathways. Furthermore, the NRF2 signaling pathway which is upstream of the ATF3-MAPKinase signaling pathway, is also regulated by the mitochondrial oxidative stress. We are the first to report differential inflammatory characteristics of transcript variants 4 (ATF3-T4) and 5 (ATF3-T5) of the stress responsive gene ATF3 in HBMECs induced by postprandial TL. Specifically, our data indicates that ATF3-T4 predominantly regulates the TL-induced brain microvascular inflammation and TNF signaling. Both siRNAs of ATF3-T4 and ATF3-T5 suppress cells apoptosis and lipotoxic brain microvascular endothelial cells. These novel signaling pathways triggered by oxidative stress-responsive transcript variants, ATF3-T4 and ATF3-T5, in the brain microvascular inflammation induced by TGRL lipolysis products may contribute to pathophysiological processes of vascular dementia.

Divergent transcriptomic signatures in response to salinity exposure in two populations of an estuarine fish.

In estuary and coastal systems, human demand for freshwater, climate change-driven precipitation variability, and extreme weather impact salinity levels, reducing connectivity between mesohaline coastal fish populations and potentially contributing to genomic divergence. We examined gill transcriptome responses to salinity in wild-caught juveniles from two populations of Sacramento splittail (Pogonichthys macrolepidotus), a species of conservation concern that is endemic to the San Francisco Estuary, USA, and the lower reaches of its tributaries. Recent extreme droughts have led to salinities above the tolerance limits for this species, creating a migration barrier between these populations, which potentially contributed to population divergence. We identified transcripts involved in a conserved response to salinity; however, the more salinity-tolerant San Pablo population had greater transcriptome plasticity (3.6-fold more transcripts responded than the Central Valley population) and a response consistent with gill remodeling after 168 hr of exposure to elevated salinity. The reorganization of the gill in response to changing osmotic gradients is a process critical for acclimation and would facilitate enhanced salinity tolerance. We detected an upregulation of receptors that control the Wnt (wingless-type) cell signaling pathway that may be required for an adaptive response to increases in salinity, patterns not observed in the relatively salinity-sensitive Central Valley population. We detected 62 single nucleotide polymorphisms (SNPs) in coding regions of 26 transcripts that differed between the populations. Eight transcripts that contained SNPs were associated with immune responses, highlighting the importance of diversity in immune gene sequences as a defining characteristic of genomic divergence between these populations. Our data demonstrate that these populations have divergent transcriptomic responses to salinity, which is consistent with observed physiological differences in salinity tolerance.

Proteome and metabolome analyses reveal differential responses in tomato -Verticillium dahliae-interactions.

Verticillium dahliae colonizes vascular tissue and causes vascular discoloration in susceptible hosts. Two well-defined races exist in V. dahliae populations from tomato and lettuce. In this study, proteins and metabolites obtained from stems of race 1-incompatible (Beefsteak) and -compatible (Early Pak) tomato cultivars were characterized. A total of 814 and 584 proteins in Beefsteak; and 456 and 637 proteins in Early Pak were identified in stem extracts of plants inoculated with races 1 and 2, respectively. A significant number of defense-related proteins were expressed in each tomato-V. dahliae interaction, as anticipated. However, phenylalanine ammonia-lyase (PAL), an important defense-associated enzyme of the phenylpropanoid pathway, in addition to remorin 1, NAD-dependent epimerase/dehydratase, and polyphenol oxidase were uniquely expressed in the incompatible interaction. Compared with the uninoculated control, significant overexpression of gene ontology terms associated with lignin biosynthesis, phenylpropanoid pathway and carbohydrate methylation were identified exclusively in the incompatible interaction. Phenolic compounds known to be involved in plant defense mechanisms were at higher levels in the incompatible relative to the compatible interactions. Based on our findings, PAL and enzymes involved defense-related secondary metabolism and the strengthening of cell walls is likely critical to confer resistance to race 1 of V. dahliae in tomato. SIGNIFICANCE: Verticillium dahliae, a soilborne fungal pathogen and a widely distributed fungal pathogen, colonizes vascular tissue and causes vascular discoloration in roots and stems, leaf wilting, and death of susceptible plant hosts. It causes billions of dollars in annual crop losses all over the world. The study focused on the proteomic and metabalomic of V. dahliae interactions (incompatible with Beefsteak and compatible with Early Pak tomato cultivars). Based on our findings, PAL and enzymes involved defense-related secondary metabolism and the strengthening of cell walls is likely critical to confer resistance to race 1 of V. dahliae in tomato.

Transcriptome Analysis of Salmonella Heidelberg after Exposure to Cetylpyridinium Chloride, Acidified Calcium Hypochlorite, and Peroxyacetic Acid.

The application of RNA sequencing in commercial poultry could facilitate a novel approach toward food safety with respect to identifying conditions in food production that mitigate transcription of genes associated with virulence and survivability. In this study, we evaluated the effects of disinfectant exposure on the transcriptomes of two field isolates of Salmonella Heidelberg (SH) isolated from a commercial broiler processing plant in 1992 and 2014. The isolates were each exposed separately to the following disinfectants commonly used in poultry processing: cetylpyridinium chloride (CPC), acidified calcium hypochlorite (aCH), and peroxyacetic acid (PAA). Exposure times were 8 s with CPC to simulate a poultry processing dipping station or 90 min with aCH and PAA to simulate the chiller tank in a poultry processing plant at 4°C. Based on comparison with a publicly available annotated SH reference genome with 5,088 genes, 90 genes were identified as associated with virulence, pathogenicity, and resistance (VPR). Of these 90 VPR genes, 9 (10.0%), 28 (31.1%), and 1 (1.1%) gene were upregulated in SH 2014 and 21 (23.3%), 26 (28.9%), and 2 (2.2%) genes were upregulated in SH 2014 challenged with CPC, aCH, and PAA, respectively. This information and previously reported MICs for the three disinfectants with both SH isolates allow researchers to make more accurate recommendations regarding control methods of SH and public health considerations related to SH in food production facilities where SH has been isolated. For example, the MICs revealed that aCH is ineffective for SH inhibition at regulatory levels allowed for poultry processing and that aCH was ineffective for inhibiting SH growth and caused an upregulation of VPR genes.

Additional Evidence for DDB2 T338M as a Genetic Risk Factor for Ocular Squamous Cell Carcinoma in Horses.

Squamous cell carcinoma (SCC) is the most common periocular cancer in horses and the second most common tumor of the horse overall. A missense mutation in damage-specific DNA-binding protein 2 (DDB2, c.1012 C>T, p.Thr338Met) was previously found to be strongly associated with ocular SCC in Haflinger and Belgian horses, explaining 76% of cases across both breeds. To determine if this same variant in DDB2 contributes to risk for ocular SCC in the Arabian, Appaloosa, and Percheron breeds and to determine if the variant contributes to risk for oral or urogenital SCC, histologically confirmed SCC cases were genotyped for the DDB2 variant and associations were investigated. Horses with urogenital SCC that were heterozygous for the DDB2 risk allele were identified in the Appaloosa breed, but a significant association between the DDB2 variant and SCC occurring at any location in this breed was not detected. The risk allele was not identified in Arabians, and no Percherons were homozygous for the risk allele. High-throughput sequencing data from six Haflingers were analyzed to ascertain if any other variant from the previously associated 483 kb locus on ECA12 was more concordant with the SCC phenotype than the DDB2 variant. Sixty polymorphisms were prioritized for evaluation, and no other variant from this locus explained the genetic risk better than the DDB2 allele (P = 3.39 × 10-17, n = 118). These data provide further support of the DDB2 variant contributing to risk for ocular SCC, specifically in the Haflinger and Belgian breeds.

Bone formation transcripts dominate the differential gene expression profile in an equine osteoporotic condition associated with pulmonary silicosis.

Osteoporosis has been associated with pulmonary silicosis in California horses exposed to soils rich in cytotoxic silica dioxide crystals, a syndrome termed silicate associated osteoporosis (SAO). The causal mechanism for the development of osteoporosis is unknown. Osteoporotic lesions are primarily located in bone marrow-rich sites such as ribs, scapula and pelvis. Gene transcription patterns within bone marrow and pulmonary lymph nodes of affected horses may offer clues to disease pathobiology. Bone marrow core and tracheobronchial lymph node tissue samples harvested postmortem from affected and unaffected horses were examined histologically and subjected to RNA sequencing (RNA-seq). Sequenced data were analyzed for differential gene expression and gene ontology. Metatranscriptomic and metagenomic assays evaluated samples for infectious agents. Thirteen of 17 differentially expressed transcripts in bone marrow were linked to bone and cartilage formation such as integrin binding bone sialoprotein (log2FC = 3.39, PFDR = 0.013) and chondroadherin (log2FC = 4.48, PFDR = 0.031). Equus caballus solute carrier family 9, subfamily A2 (log2FC = 3.77, PFDR = 0.0034) was one of the four differentially expressed transcripts linked to osteoclast activity. Osteoblasts were hyperplastic and hypertrophic in bone marrow from affected horses. Biological pathways associated with skeletal morphogenesis were significantly enriched in affected horses. The 30 differentially expressed genes in affected lymph nodes were associated with inflammatory responses. Evidence of infectious agents was not found. The SAO affected bone marrow molecular signature demonstrated increased transcription and heightened activation of osteoblasts. Increased osteoblastic activity could be part of the pathological mechanism for osteoporosis or a compensatory response to the accelerated osteolysis. Transcriptome data offer gene targets for inquiries into the role of osteocytes and osteoblasts in SAO pathogenesis. Viral or bacterial infectious etiology in SAO is less likely based on metatranscriptomic and metagenomic data but cannot be completely ruled out.