582 research outputs found
The USDA cucumber (Cucumis sativus L.) collection: genetic diversity, population structure, genome-wide association studies, and core collection development
No full textCucumber: Core collection for future crop improvement Genetic analysis of the US cucumber collection provides valuable insights into the plant’s diversity and generates a core resource for future research. Breeding crop plants requires in-depth understanding of plant genetics and the identification of key genes that enhance traits such as disease and stress resistance. Zhangjun Fei at the Boyce Thompson Institute in Ithaca, Rebecca Grumet at Michigan State University, and co-workers across the US used high-throughput genetic sequencing to characterize the 1,234 cucumber accessions held by the US National Plant Germplasm System. The team uncovered 23,000 specific genome variations that revealed the genetic diversity, population structure and genetic differentiation of the cucumber. They identified genome regions associated with 13 traits important to cultivation and selected 395 accessions covering 96 percent of the collection’s genetic variation to make a publicly accessible core of data
Transcriptomics-based screen for genes induced by flagellin and repressed by pathogen effectors identifies a cell wall-associated kinase involved in plant immunity
No full textBackground: Microbe-associated molecular patterns, such as those present in bacterial flagellin, are powerful inducers of the innate immune response in plants. Successful pathogens deliver virulence proteins, termed effectors, into the plant cell where they can interfere with the immune response and promote disease. Engineering the plant immune system to enhance disease resistance requires a thorough understanding of its components. Results: We describe a high-throughput screen, using RNA sequencing and virus-induced gene silencing, to identify tomato genes whose expression is enhanced by the flagellin microbe-associated molecular pattern flgII-28, but reduced by activities of the Pseudomonas syringae pv. tomato (Pst) type III effectors AvrPto and AvrPtoB. Gene ontology terms for this category of Flagellin-induced repressed by effectors (FIRE) genes showed enrichment for genes encoding certain subfamilies of protein kinases and transcription factors. At least 25 of the FIRE genes have been implicated previously in plant immunity. Of the 92 protein kinase-encoding FIRE genes, 33 were subjected to virus-induced gene silencing and their involvement in pattern-triggered immunity was tested with a leaf-based assay. Silencing of one FIRE gene, which encodes the cell wall-associated kinase SlWAK1, compromised the plant immune response resulting in increased growth of Pst and enhanced disease symptoms. Conclusions: Our transcriptomic approach identifies FIRE genes that represent a pathogen-defined core set of immune-related genes. The analysis of this set of candidate genes led to the discovery of a cell wall-associated kinase that participates in plant defense. The FIRE genes will be useful for further elucidation of the plant immune system.Fil: Rosli, Hernan Guillermo. Boyce Thompson Institute for Plant Research; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Zhen, Yi. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Pombo, Marina Alejandra. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Zhong, Silin. Boyce Thompson Institute for Plant Research; Estados Unidos. University of Hong Kong; Hong KongFil: Bombarely, Aureliano. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Fei, Zhangjun. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Collmer, Alan. Cornell University; Estados Unidos. King Abdulaziz University; Estados UnidosFil: Martin, Gregory B.. Boyce Thompson Institute for Plant Research; Estados Unidos. Cornell University; Estados Unidos. King Abdulaziz University; Estados Unido
Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem
No full textThe agronomical relevant tomato-Pseudomonas syringae pv. tomato pathosystem is widely used to explore and understand the underlying mechanisms of the plant immune response. Transcript abundance estimation, mainly through reverse transcription-quantitative PCR (RT-qPCR), is a common approach employed to investigate the possible role of a candidate gene in certain biological process under study. The accuracy of this technique relies heavily on the selection of adequate reference genes. Initially, genes derived from other techniques (such as Northern blots) were used as reference genes in RT-qPCR experiments, but recent studies in different systems suggest that many of these genes are not stably expressed. The development of high throughput transcriptomic techniques, such as RNA-seq, provides an opportunity for the identification of transcriptionally stable genes that can be adopted as novel and robust reference genes. Here we take advantage of a large set of RNA-seq data originating from tomato leaves infiltrated with different immunity inducers and bacterial strains. We assessed and validated 9 genes that are much more stable than two traditional reference genes. Specifically, ARD2 and VIN3 were the most stably expressed genes and consequently we propose they be adopted for RT-qPCR experiments involving this pathosystem.Fil: Pombo, Marina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; ArgentinaFil: Zheng, Yi. Boyce Thompson Institute for Plant Research; Estados UnidosFil: Fei, Zhangjun. Boyce Thompson Institute for Plant Research; Estados Unidos. United States Department of Agriculture; Estados UnidosFil: Martin, Gregory B.. Boyce Thompson Institute for Plant Research; Estados Unidos. Cornell University; Estados UnidosFil: Rosli, Hernan Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentin
Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato- Pseudomonas pathosystem
No full textThe agronomical relevant tomato-Pseudomonas syringae pv. tomato pathosystem is widely used to explore and understand the underlying mechanisms of the plant immune response. Transcript abundance estimation, mainly through reverse transcription-quantitative PCR (RT-qPCR), is a common approach employed to investigate the possible role of a candidate gene in certain biological process under study. The accuracy of this technique relies heavily on the selection of adequate reference genes. Initially, genes derived from other techniques (such as Northern blots) were used as reference genes in RT-qPCR experiments, but recent studies in di erent systems suggest that many of these genes are not stably expressed. The development of high throughput transcriptomic techniques, such as RNA-seq, provides an opportunity for the identi cation of transcriptionally stable genes that can be adopted as novel and robust reference genes. Here we take advantage of a large set of RNA-seq data originating from tomato leaves in ltrated with di erent immunity inducers and bacterial strains. We assessed and validated 9 genes that are much more stable than two traditional reference genes. Speci cally, ARD2 and VIN3 were the most stably expressed genes and consequently we propose they be adopted for RT- qPCR experiments involving this pathosystem.Fil: Pombo, Marina Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; ArgentinaFil: Zheng, Yi. Cornell University; Estados UnidosFil: Fei, Zhangjun. Cornell University; Estados UnidosFil: Martin, Gregory. Cornell University; Estados UnidosFil: Rosli, Hernan Guillermo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentin
Use of homologous and heterologous gene expression profiling tools to characterize transcription dynamics during apple fruit maturation and ripening
No full textAbstract Background Fruit development, maturation and ripening consists of a complex series of biochemical and physiological changes that in climacteric fruits, including apple and tomato, are coordinated by the gaseous hormone ethylene. These changes lead to final fruit quality and understanding of the functional machinery underlying these processes is of both biological and practical importance. To date many reports have been made on the analysis of gene expression in apple. In this study we focused our investigation on the role of ethylene during apple maturation, specifically comparing transcriptomics of normal ripening with changes resulting from application of the hormone receptor competitor 1-Methylcyclopropene. Results To gain insight into the molecular process regulating ripening in apple, and to compare to tomato (model species for ripening studies), we utilized both homologous and heterologous (tomato) microarray to profile transcriptome dynamics of genes involved in fruit development and ripening, emphasizing those which are ethylene regulated. The use of both types of microarrays facilitated transcriptome comparison between apple and tomato (for the later using data previously published and available at the TED: tomato expression database) and highlighted genes conserved during ripening of both species, which in turn represent a foundation for further comparative genomic studies. The cross-species analysis had the secondary aim of examining the efficiency of heterologous (specifically tomato) microarray hybridization for candidate gene identification as related to the ripening process. The resulting transcriptomics data revealed coordinated gene expression during fruit ripening of a subset of ripening-related and ethylene responsive genes, further facilitating the analysis of ethylene response during fruit maturation and ripening. Conclusion Our combined strategy based on microarray hybridization enabled transcriptome characterization during normal climacteric apple ripening, as well as definition of ethylene-dependent transcriptome changes. Comparison with tomato fruit maturation and ethylene responsive transcriptome activity facilitated identification of putative conserved orthologous ripening-related genes, which serve as an initial set of candidates for assessing conservation of gene activity across genomes of fruit bearing plant species.</p
EVALUATING TRANSCRIPTOME CHANGES IN SWEET POTATO DURING VIRAL PATHOGEN INFECTION
No full text117 pagesSweet potato (Ipomoea batatas) ranks among the most important crops in the world and provides nutritional and economic sustainability for subsistence farmers in Sub-Saharan Africa. Its production is mainly constrained by Sweet potato virus disease (SPVD) caused by the coinfection by two positive-sense single-stranded RNA viruses, Sweet potato chlorotic stunt virus and Sweet potato feathery mottle virus. Current understanding of sweet potato responses to these viruses at the molecular level remains very limited. In this study, we performed deep transcriptome sequencing in three sweet potato cultivars with varying degrees of viral resistance, coupled with comprehensive and integrated analytical approaches, to identify biological pathways that contribute to both general and specific host responses to these important viral pathogens. We found that much of the antiviral response was effector-triggered immunity (ETI) specific, and that the expression patterns (both timing and magnitude) of this response were dependent on each cultivar’s resistance to SPVD
UNDERSTANDING INSECT MIDGUT RECEPTORS FOR BT PROTEIN CRY1AC AND MECHANISM OF RESISTANCE TO CRY1AC IN THE CABBAGE LOOPER, TRICHOPLUSIA NI
No full text226 pagesThe successful deployment of insecticidal proteins from Bacillus thuringiensis (Bt) via genetically engineered crops has revolutionized pest management practices in agriculture. However, the development of insect resistance to Bt toxins threatens the sustainable application of Bt-biotechnology. Current understanding of Bt resistance has indicated that resistance to Bt in insects is complex, involving multiple midgut proteins that serve as receptors for Bt toxins in the intoxication pathways. In this dissertation, I studied the genetic basis of resistance to Bt protein Cry1Ac in a greenhouse-evolved resistant strain of the cabbage looper, Trichoplusia ni. Using genetic and molecular approaches, I studied the genetic association of altered APN (Aminopeptidase N) expression with the ABCC2 (ABC transporter C2) and the association of Cry1Ac resistance with APNs. The results indicated that the downregulated APN1 expression in resistant T. ni and low expression of APN6 in susceptible T. ni were associated with mutations in APN1 and APN6 genes but were not with ABCC2. The high-level resistance to Cry1Ac in T. ni is associated with both ABCC2 and APN1 mutations and additional factors to be identified. The functional roles of putative Cry1Ac receptors ABCC2, ALP (alkaline phosphatase), APN1, and midgut cadherin (CAD) in larval susceptibility to Cry1Ac were systematically examined, using a series of gene knockout T. ni mutant strains. ABCC2 was confirmed to be a major receptor for Cry1Ac in T. ni, but knockout mutations in the ALP, APN, and CAD receptors resulted in none to low resistance in T. ni. Results also indicated additional resistance genes to be identified. Finally, I studied the role of carbohydrate moieties in the mode of action of Cry1Ac. The interaction of Cry1A with the midgut and Cry1Ac toxicity in larvae were analyzed in vitro and in vivo, using a carbohydrate binding chemical, Calcofluor. The results demonstrated that carbohydrate moieties play critically important roles in the functional specific binding of the toxin to the midgut receptors in the pathway of toxicity. Overall, the findings from this thesis research advanced our understanding of the mode of action of Cry proteins and mechanisms of insect resistance to Cry proteins
Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit
No full textNEW INSIGHTS INTO THE PHYSIOLOGY, BIOSYNTHESIS, AND MOLECULAR CONTROLS OF ORGANIC ACIDS AND POLYPHENOLS IN CIDER APPLES
No full text196 pagesThe organic acids and polyphenols in apple (Malus ×domestica) juice are responsible for hard cider flavor, aroma, color, and microbial stability. The second chapter of this dissertation describes how the malic acid marker Ma1 was able to categorize 217 cider apple cultivars into low (5.8 g·L-1) acidity groups. Triploid cultivars had a significant 0.36 g·L-1 greater titratable acidity than diploid cultivars (P = 0.0111). The third and fourth chapters focused on the effect of crop density and early tree shading on polyphenol development in cider apples respectively to understand source-sink relationships and explain the year-to-year variation in polyphenol content. There was a significant increase in the concentrations of most polyphenol compounds, including monomeric and oligomeric proanthocyanidin compounds in the low crop density treatment (5 fruit/cm2 trunk cross-sectional area) compared to the unthinned control (P < 0.0100). Transcriptome profiling through RNA sequencing indicated the critical genes involved in hydroxylation, methylation, and glycosylation in the phenylpropanoid pathway were upregulated in the low crop density treatment at 27 DAFB and 81 DAFB, which corresponded with increased concentration of phenylpropanoids. Specifically, there was a significant increase in the expression of the gene encoding anthocyanidin reductase (catalyzes the production of epicatechin) in the low crop density treatment at 27 and 81 days after full bloom (P < 0.0100). In Chapter 4, carbohydrate stress applied through early tree shading (1-5 weeks after full bloom) reduced phenolic acids and quercetin glycoside concentrations at harvest in the 60-tree shade treatment (60% of photosynthetically active radiation blocked) in comparison to the unshaded control, with minimal impact on production of procyanidin monomers and oligomers in cider apples. Fruit shaded trees were not significantly different from the control. This dissertation elucidates a new marker-based acidity classification system allowing for cultivar comparisons across geographical, seasonal, and horticultural considerations; illustrates the differing polyphenol accumulation patterns in the peel and flesh tissue; provides compelling evidence for a positive source-sink relationship with polyphenol development in cider apples; and lists transcription factors that could possibly be involved in the polyphenol production in cider apples
USING HIGH-THROUGHPUT TECHNOLOGIES TO ADVANCE OUR UNDERSTANDING ON POTATO VIRUS Y INFECTIONS
No full textSupplemental file(s) description: Supplemental Table 1, Supplemental Table 2, Supplemental Table 3, Supplemental Table 4Potato virus Y (PVY) is a major virus pathogen of potato worldwide. Surveys indicate that re-combinant strains of PVY have emerged in recent years to predominate in the U.S. potato crop and that the genetic diversity among and within PVY strains is prodigious. Vegetative propaga-tion of potato via tubers allows PVY to survive year to year and to be transported over long dis-tances. Whereas, aphids are primarily responsible for spread of PVY within the crop and over regional distances. Furthermore, the tuber necrotic strain (PVYNTN) is associated with potato tuber necrotic ringspot disease (PTNRD), a tuber deformity that negatively impacts tuber quali-ty, marketability, and poses a serious threat to seed and commercial potato production world-wide. To map loci that influence tuber and foliar symptoms in potatoes infected with PVYNTN and the length of tuber dormancy, two potato populations were genotyped with a potato SNP chip. QTL analyses revealed major-effect QTLs in a Waneta x Pike cross for mosaic on chromosomes 4 and 5, and for PTNRD and for foliar-necrosis symptoms on chromosomes 4 and 5, respective-ly. QTLs for dormancy were detected on chromosomes 2, 3, 5, 6, and 8, in a Waneta x Superior cross. Locating QTLs associated with PVY-related symptoms and tuber dormancy provides a framework for breeders to develop varieties with resistance to multiple PVY-symptoms and to manipulate tuber dormancy length. Illumina high-throughput sequencing was used to study the quasispecies diversity of 15 isolates representing seven different PVY strains and to investigate how transmission modes (insect and mechanical) are contributing to the evolution and diversification of PVY. Eight were PVYN isolates and six of those came from the same geographic region in two different years. A con-sensus sequence, without indels or insertions, was successfully extracted from the sequenced reads of each isolate after being mapped to the strain reference genome. PVYN had a higher population genetic diversity than any other strain evaluated and the population genetic diversi-ty of the PVYN isolates, differed between collection years and sites. We found that the popula-tion diversity of PVY varies by the virus strain but doesn’t differ among transmission modes. Our data suggest that each transmission mode exerts unique selection pressures on the virus population and allows different mutations to accumulate and become fixed. Knowledge of how rapidly PVY can evolve and of the factors driving PVY quasispecies diversity could be used to enhance the efficiency of PVY management practices in potato fields
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