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Abstract Background Desirable apple varieties are clonally propagated by grafting vegetative scions onto rootstocks. Rootstocks influence many phenotypic traits of the scion, including resistance to pathogens such as Erwinia amylovora, which causes fire blight, the most serious bacterial disease of apple. The purpose of the present study was to quantify rootstock-mediated differences in scion fire blight susceptibility and to identify transcripts in the scion whose expression levels correlated with this response. Results Rootstock influence on scion fire blight resistance was quantified by inoculating three-year old, orchard-grown apple trees, consisting of 'Gala' scions grafted to a range of rootstocks, with E. amylovora. Disease severity was measured by the extent of shoot necrosis over time. 'Gala' scions grafted to G.30 or MM.111 rootstocks showed the lowest rates of necrosis, while 'Gala' on M.27 and B.9 showed the highest rates of necrosis. 'Gala' scions on M.7, S.4 or M.9F56 had intermediate necrosis rates. Using an apple DNA microarray representing 55,230 unique transcripts, gene expression patterns were compared in healthy, un-inoculated, greenhouse-grown 'Gala' scions on the same series of rootstocks. We identified 690 transcripts whose steady-state expression levels correlated with the degree of fire blight susceptibility of the scion/rootstock combinations. Transcripts known to be differentially expressed during E. amylovora infection were disproportionately represented among these transcripts. A second-generation apple microarray representing 26,000 transcripts was developed and was used to test these correlations in an orchard-grown population of trees segregating for fire blight resistance. Of the 690 transcripts originally identified using the first-generation array, 39 had expression levels that correlated with fire blight resistance in the breeding population. Conclusions Rootstocks had significant effects on the fire blight susceptibility of 'Gala' scions, and rootstock-regulated gene expression patterns could be correlated with differences in susceptibility. The results suggest a relationship between rootstock-regulated fire blight susceptibility and sorbitol dehydrogenase, phenylpropanoid metabolism, protein processing in the endoplasmic reticulum, and endocytosis, among others. This study illustrates the utility of our rootstock-regulated gene expression data sets for candidate trait-associated gene data mining.

Background: Adolescent sensitivity to alcohol is influenced by genetic background. Data from our laboratory suggested that adolescent C57BL/6J and DBA/2J inbred mice differed in susceptibility to alcohol-induced deficits in dorsal hippocampus-dependent contextual fear learning., Methods: To investigate the biological underpinnings of this strain difference, we examined dorsal hippocampus gene expression using RNA-sequencing after alcohol or saline administration followed by Pavlovian fear conditioning across male and female C57BL/6J and DBA/2J adolescents., Results: Strains exhibited dramatic differences in dorsal hippocampus gene expression. Specifically, C57BL/6J and DBA/2J strains differed by 3526 transcripts in males and 2675 transcripts in females. We identified pathways likely to be involved in mediating alcohol's effects on learning, including networks associated with Chrna7, a gene encoding the nicotinic cholinergic receptor alpha 7 subunit, and Fmr1, a gene encoding the fragile X messenger ribonucleoprotein., Conclusions: These findings provide insight into the mechanisms underlying strain differences in alcohol's effects on learning and suggest that different biological networks are recruited for learning based on genetics, sex, and alcohol exposure., (© 2024 The Author(s). Alcohol, Clinical and Experimental Research published by Wiley Periodicals LLC on behalf of Research Society on Alcohol.)

Background: Theobroma cacao, the cocoa tree, is a tropical crop grown for its highly valuable cocoa solids and fat which are the basis of a 200-billion-dollar annual chocolate industry. However, the long generation time and difficulties associated with breeding a tropical tree crop have limited the progress of breeders to develop high-yielding disease-resistant varieties. Development of marker-assisted breeding methods for cacao requires discovery of genomic regions and specific alleles of genes encoding important traits of interest. To accelerate gene discovery, we developed a gene atlas composed of a large dataset of replicated transcriptomes with the long-term goal of progressing breeding towards developing high-yielding elite varieties of cacao., Results: We describe the creation of the Cacao Transcriptome Atlas, its global characterization and define sets of genes co-regulated in highly organ- and temporally-specific manners. RNAs were extracted and transcriptomes sequenced from 123 different tissues and stages of development representing major organs and developmental stages of the cacao lifecycle. In addition, several experimental treatments and time courses were performed to measure gene expression in tissues responding to biotic and abiotic stressors. Samples were collected in replicates (3-5) to enable statistical analysis of gene expression levels for a total of 390 transcriptomes. To promote wide use of these data, all raw sequencing data, expression read mapping matrices, scripts, and other information used to create the resource are freely available online. We verified our atlas by analyzing the expression of genes with known functions and expression patterns in Arabidopsis (ACT7, LEA19, AGL16, TIP13, LHY, MYB2) and found their expression profiles to be generally similar between both species. We also successfully identified tissue-specific genes at two thresholds in many tissue types represented and a set of genes highly conserved across all tissues., Conclusion: The Cacao Gene Atlas consists of a gene expression browser with graphical user interface and open access to raw sequencing data files as well as the unnormalized and CPM normalized read count data mapped to several cacao genomes. The gene atlas is a publicly available resource to allow rapid mining of cacao gene expression profiles. We hope this resource will be used to help accelerate the discovery of important genes for key cacao traits such as disease resistance and contribute to the breeding of elite varieties to help farmers increase yields., (© 2024. The Author(s).)

We report the complete 4,352,172-bp genome sequence of Mycobacterium orygis strain 51145 assembled into a single circular chromosome. Comparative genomic analyses with other lineages of the Mycobacterium tuberculosis complex can provide insights into the biology, evolution, and epidemiology of this important group of pathogenic mycobacteria., (Copyright © 2021 Rufai et al.)

Background: Apple tree breeding is slow and difficult due to long generation times, self-incompatibility, and complex genetics. The identification of molecular markers linked to traits of interest is a way to expedite the breeding process. In the present study, we aimed to identify genes whose steady-state transcript abundance was associated with inheritance of specific traits segregating in an apple (Malus × domestica) rootstock F1 breeding population, including resistance to powdery mildew (Podosphaera leucotricha) disease and woolly apple aphid (Eriosoma lanigerum)., Results: Transcription profiling was performed for 48 individual F1 apple trees from a cross of two highly heterozygous parents, using RNA isolated from healthy, actively-growing shoot tips and a custom apple DNA oligonucleotide microarray representing 26,000 unique transcripts. Genome-wide expression profiles were not clear indicators of powdery mildew or woolly apple aphid resistance phenotype. However, standard differential gene expression analysis between phenotypic groups of trees revealed relatively small sets of genes with trait-associated expression levels. For example, thirty genes were identified that were differentially expressed between trees resistant and susceptible to powdery mildew. Interestingly, the genes encoding twenty-four of these transcripts were physically clustered on chromosome 12. Similarly, seven genes were identified that were differentially expressed between trees resistant and susceptible to woolly apple aphid, and the genes encoding five of these transcripts were also clustered, this time on chromosome 17. In each case, the gene clusters were in the vicinity of previously identified major quantitative trait loci for the corresponding trait. Similar results were obtained for a series of molecular traits. Several of the differentially expressed genes were used to develop DNA polymorphism markers linked to powdery mildew disease and woolly apple aphid resistance., Conclusions: Gene expression profiling and trait-associated transcript analysis using an apple F1 population readily identified genes physically linked to powdery mildew disease resistance and woolly apple aphid resistance loci. This result was especially useful in apple, where extreme levels of heterozygosity make the development of reliable DNA markers quite difficult. The results suggest that this approach could prove effective in crops with complicated genetics, or for which few genomic information resources are available.

An antibody microarray was developed to detect the "top six" non-O157 serogroups, O26, O45, O103, O111, O121, and O145 of Shiga toxin-producing Escherichia coli (STEC), that have been declared as adulterant in meat by the Food Safety and Inspection Service of the United States Department of Agriculture. The sensitivity of the array was 10(5)CFU and the limit of detection of each serogroup in artificially inoculated ground beef was 1-10 CFU following 12h of enrichment. Optimal concentrations of antibodies for printing and labeling and bacterial dilutions for binding to the antibodies were assessed. The array utilized a minimal amount of antibodies and other reagents and may be utilized for screening of multiple target O groups of STEC in parallel, directly from enriched samples in less than 3h. Furthermore, the antibody array provides the flexibility to include other O serogroups of E. coli and may be adopted for high throughput screening. The method is potentially applicable to detect the pathogenic STEC O groups of E. coli in meat and other food, thus improving food safety and public health., (Copyright © 2013 Elsevier B.V. All rights reserved.)

To transform undergraduate biology education, faculty need to provide opportunities for students to engage in the process of science. The rise of research approaches using next-generation (NextGen) sequencing has been impressive, but incorporation of such approaches into the undergraduate curriculum remains a major challenge. In this paper, we report proceedings of a National Science Foundation-funded workshop held July 11-14, 2011, at Juniata College. The purpose of the workshop was to develop a regional research coordination network for undergraduate biology education (RCN/UBE). The network is collaborating with a genome-sequencing core facility located at Pennsylvania State University (University Park) to enable undergraduate students and faculty at small colleges to access state-of-the-art sequencing technology. We aim to create a database of references, protocols, and raw data related to NextGen sequencing, and to find innovative ways to reduce costs related to sequencing and bioinformatics analysis. It was agreed that our regional network for NextGen sequencing could operate more effectively if it were partnered with the Genome Consortium for Active Teaching (GCAT) as a new arm of that consortium, entitled GCAT-SEEK(quence). This step would also permit the approach to be replicated elsewhere.

Longitudinal bone growth depends upon the execution of an intricate series of cellular activities by epiphyseal growth plate chondrocytes. In order to better understand these coordinated events, microarray analysis was used to compare gene expression in chondrocytes isolated from the proliferative and hypertrophic zones of the avian growth plate. RT-PCR was used to confirm the identity of a select number of genes. The expression of 745 genes was found to differ 3-fold or greater at the 0.05 level of probability. Transferrin was the most highly up-regulated (321-fold) gene associated with chondrocyte hypertrophy. Immunohistochemistry localized this peptide adjacent to the penetrating blood vessels in the growth plate of 3-week-old chicks. Fibulin, OC-116, DMP-1 and PHEX were among the expanded number of genes associated with extracellular matrix metabolism. The presence of NELL2, ATOH8 and PLEXIN suggests a neuronal involvement in growth plate physiology. In addition, the expression of a large number of genes associated with angiogenesis and cellular stress was up-regulated. These processes are important to the physiology and survival of chondrocytes in the unique and stressful environment of the epiphyseal growth plate., (Copyright 2009 Elsevier Inc. All rights reserved.)

Components of the insulin-like growth factor (IGF) system were investigated in chondrocytes isolated from the avian growth plate. The genes for IGF-I, IGF-II, type 1 IGF receptor (IGF-R), IGF binding protein-2 (IGFBP-2), IGFBP-3, IGFBP-5 and IGFBP-7 were found to be expressed in both proliferative and hypertrophic chondrocytes. The expression of IGF-II in proliferative chondrocytes was extremely high relative to IGF-I. Although IGF-I expression was significantly increased in hypertrophic chondrocytes, the level was still low relative to IGF-II. In cell culture, IGF-I stimulated proteoglycan synthesis and increased the expression of Indian hedgehog (Ihh) and type X collagen, markers of chondrocyte differentiation. IGF-II was found to be equally efficacious in stimulating proteoglycan biosynthesis. These observations suggest that IGF-II may play a significant role in avian growth plate physiology, which is consistent with several reports on mammalian endochondral bone growth.

The purpose of this investigation was to study the expression of type X collagen in the hen's oviduct. Type X collagen is a short-chain collagen that is present in the fibers of eggshell membranes, and there is evidence to suggest that it contributes to structural integrity. In situ hybridization and Northern blot analysis were used to study the expression of this important matrix constituent. The results demonstrated that gene expression was predominantly in the tubular gland cells of the isthmus segment of the oviduct. In contrast to observations with other matrix proteins, such as parathyroid hormone-related peptide and osteopontin, gene expression did not fluctuate with the position of the egg in the oviduct.

Parathyroid hormone-related peptide (PTHrP) has been shown to be essential for normal endochondral bone formation. Along with Indian hedgehog (Ihh), it forms a paracrine regulatory loop that governs the pace of chondrocyte differentiation. However, the source of PTHrP for this regulatory loop is not clear. While one hypothesis has suggested the periarticular perichondrium as the source of PTHrP for growth plate regulation, other data utilizing immunohistochemistry and in situ hybridization would indicate that growth plate chondrocytes themselves are the source of this peptide. The data described in this report supports the view that postnatal growth plate chondrocytes have the ability to synthesize this important regulatory peptide. Immunohistochemistry of tissue sections showed that PTHrP protein was evident throughout the chick epiphysis. PTHrP was seen in chondrocytes in the periarticular perichondrium, the perichondrium adjacent to the growth plate, the prehypertrophic zone of the growth plate, and the hypertrophic zone of the growth plate. However, cells in the proliferative zone, as well as some chondrocytes in the deeper layers of articular cartilage were predominantly negative for PTHrP. PTHrP was detected by Western blotting as a band of 16,400 Da in extracts from hypertrophic chondrocytes, but not from proliferative cells. RT-PCR detected PTHrP mRNA in both proliferative and hypertrophic growth plate chondrocytes, as well as in articular chondrocytes. PTH/PTHrP receptor mRNA was detected by Northern blotting in growth plate, but not articular chondrocytes. Thus, we conclude that most of the PTHrP present in the epiphyseal growth plate of the juvenile chick originates in the growth plate itself. Furthermore, the presence of large amounts of PTHrP protein in the hypertrophic zone supports the concept that PTHrP has other functions in addition to regulating chondrocyte differentiation., (Copyright 2001 Wiley-Liss, Inc.)

Tibial dyschondroplasia (TD) is a skeletal deformity associated with rapid growth in a number of avian species. The disease is the result of a disruption in the cascade of events that occur in the epiphyseal growth plate. Whereas the incidence of TD is susceptible to genetic selection, no specific genetic defect has been identified. Although there are extensive data describing the morphological and biochemical characteristics of the lesion, the mechanism of lesion formation is unknown. However, naturally occurring or induced genetic mutations in other species can provide important clues to possible mechanisms responsible for lesion development. Disruption of normal chondrocyte differentiation by constitutive activation of the parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor, inactivation of the fibroblast growth factor receptor-3 (FGFR-3) receptor, and blocking vascular endothelial growth factor (VEGF) signaling all result in lesions that resemble TD. Impairment of vascular penetration due to the ablation of matrix metalloproteinase-9 (MMP-9) or tartrate-resistant acid phosphatase (TRAP) activity also results in similar cartilage abnormalities. We have integrated these observations with our current knowledge of TD to describe a hypothesis for the sequence of events responsible for the development of tibial dyschondroplastic lesions.

When utilizing streptavidin systems with Western blots of chondrocyte, osteoblast and osteoclast lysates, proteins of the molecular weights 116 kDa, 75 kDa and 67 kDa were observed to be bound by streptavidin alone. Streptavidin binding could not be blocked by pre-incubation with an RGD containing peptide. The same proteins were bound by ExtrAvidin which lacks the RGD sequence present in streptavidin. Pre-incubation with free biotin completely abolished the binding of both streptavidin and ExtrAvidin. The three proteins observed are believed to be the biotin containing carboxylases: pyruvate carboxylase, 3-methylcrotonyl carboxylase, and propionyl carboxylase. The findings of this study underscore the need to apply vigorous controls to distinguish between endogenous biotinylated proteins and biotin used as a means to amplify avidin detection systems since a wide variety of proteins with relevance to bone and cartilage biology have molecular weights similar to the biotin carboxylases., (Copyright 1998 Academic Press.)

Tibialdyschondroplasia (TD) is a disease characterized by the formation of an avascular, non-mineralized lesion along the mature face of the epiphyseal growth plate in rapidly growing chickens. In the normal growth plate, cells progress from a proliferative phase to hypertrophy where the tissue is vascularized and replaced by trabecular bone. In TD, cells apparently cease their development early in the transition to hypertrophy. These diseased cells are not removed by vascularization nor does mineralization occur. The resulting lesion increases in size as proliferative cells continue to divide in the absence of removal and replacement of cartilage by bone. This laboratory has previously reported that cells of the TD lesion have the morphological appearance of necrotic cells or in some cases apoptotic cells. In this study we examine in more detail the status of cells comprising the TD lesion using molecular techniques. Genomic DNA isolated from cells of severe TD lesions show the nucleosomal laddering indicative of apoptosis, while DNA isolated from proliferative and hypertrophic cells does not. This result was confirmed by the use of the Cell Death Detection ELISA which shows quantitatively that cells from severe TD lesions contain nearly twice as many nucleosomal fragments as cells from the hypertrophic zone while proliferative chondrocytes do not have significant fragmentation. In situ examination of the epiphyseal growth plate with terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) clearly shows that the cells of the severe TD lesion are apoptotic. Cells from smaller lesions are stained to a lesser extent or not at all by TUNEL. We believe that the apoptosis seen in TD is a secondary effect of the disease and not its primary cause.

Basic fibroblast growth factor (bFGF) is a permissive mitogen for cultured chondrocytes and has been localized in the specific zones of the epiphyseal growth plate. In this study, we demonstrate that bFGF present in cartilage originates from within the cellular constituents of this tissue. Utilizing reverse transcription coupled to the polymerase chain reaction (PCR), bFGF mRNA was found in extracts of cartilage tissue. Immunocytochemical studies revealed that bFGF was present intracellularly in freshly isolated proliferative chondrocytes and in the extracellular matrix (ECM) after 24 h of culture. Western blot analysis of protein extracts from isolated proliferative chondrocytes identified a bFGF immunoreactive species with a molecular weight of approximately 18 kDa. In situ hybridization confirmed the presence of bFGF mRNA in freshly isolated proliferative chondrocytes. The bFGF in the ECM seemed to be sequestered and not available for biological activity, since these cells still required exogenous bFGF for cell proliferation. This sequestered bFGF could be released to stimulate cell proliferation when cultures were treated with plasmin, a proteolytic enzyme. These data support the hypothesis that bFGF is synthesized by chondrocytes and functions as an autocrine/paracrine mitogen via its deposition into the ECM with subsequent release from the ECM of cartilage being a critical step in biological activity. In addition, the study provides further evidence that locally produced bFGF plays an important role in normal growth and development of cartilage tissue.