Genomics-based Approaches Research Articles

Identification, validation and high-throughput genotyping of transcribed gene SNPs in cassava

The availability of genomic resources can facilitate progress in plant breeding through the application of advanced molecular technologies for crop improvement. This is particularly important in the case of less researched crops such as cassava, a staple and food security crop for more than 800 million people. Here, expressed sequence tags (ESTs) were generated from five drought stressed and well-watered cassava varieties. Two cDNA libraries were developed: one from root tissue (CASR), the other from leaf, stem and stem meristem tissue (CASL). Sequencing generated 706 contigs and 3,430 singletons. These sequences were combined with those from two other EST sequencing initiatives and filtered based on the sequence quality. Quality sequences were aligned using CAP3 and embedded in a Windows browser called HarvEST:Cassava which is made available. HarvEST:Cassava consists of a Unigene set of 22,903 quality sequences. A total of 2,954 putative SNPs were identified. Of these 1,536 SNPs from 1,170 contigs and 53 cassava genotypes were selected for SNP validation using Illumina's GoldenGate assay. As a result 1,190 SNPs were validated technically and biologically. The location of validated SNPs on scaffolds of the cassava genome sequence (v.4.1) is provided. A diversity assessment of 53 cassava varieties reveals some sub-structure based on the geographical origin, greater diversity in the Americas as opposed to Africa, and similar levels of diversity in West Africa and southern, eastern and central Africa. The resources presented allow for improved genetic dissection of economically important traits and the application of modern genomics-based approaches to cassava breeding and conservation.

The Evolution of Sex-Related Traits and Genes

The Evolution of Sex-Related Traits and Genes

What can the Aspergillus flavus genome offer to mycotoxin research?

The genomic study of filamentous fungi has made significant advances in recent years, and the genomes of several species in the genus Aspergillus have been sequenced, including Aspergillus flavus. This ubiquitous mold is present as a saprobe in a wide range of agricultural and natural habits, and can function as an opportunistic animal and plant pathogen. A. flavus produces many secondary metabolites including aflatoxins, aflatrem and cyclopiazonic acid. In this chapter, our main focus is on the current status of the genomics of A. flavus as well as on the potential applications of genomics-based approaches to understanding mycotoxin production and fungal pathogenicity. It is hoped that the results of A. flavus genomics and functional genomics studies will empower researchers to find effective controlling strategies to eliminate mycotoxin contamination and to yield a safer and more abundant food and feed supply.

Improvement of crop protection against greenbug using the worldwide sorghum germplasm collection and genomics-based approaches

Successful development of new sorghum cultivars and hybrids to ensure sustainable production depends largely on the availability of genetic resources with desirable traits such as pest resistance. Our recent research has focused on improvement of crop protection against greenbugs using the worldwide germplasm collection and genomics-based approaches. First, we conducted the systematic evaluation of a worldwide germplasm collection in order to identify new sources of greenbug resistance. Twenty-one resistant lines were identified, which offered new sources of resistance to sorghum breeding. Molecular markers used to assess the genetic diversity among those resistant lines suggested relatively diverse resistant sources in the sorghum germplasm collection. More recently, a mapping project was executed to associate the resistance genes with sorghum chromosomes. The mapping data indicated one major and a minor quantitative trait loci reside on chromosome 9 and are responsible for resistance to greenbug. In addition, cDNA microarrays were used to monitor greenbug-induced gene expression in sorghum plants. This study has developed a transcriptional profile for sorghum in response to greenbug attack, which provides us with useful molecular information for discovery of greenbug resistance genes and a better understanding of the genetic mechanisms controlling host defences in sorghum.

Progress in Mining the Human Proteome for Disease Applications

Currently available technologies allow in-depth analysis of multiple facets of the proteome that have clinical relevance and that complement current genomics-based approaches. Although some progress has been made in our knowledge of the human proteome in health and in disease, there is an urgent need to chart a coherent road map with clearly defined milestones to guide proteomics efforts. Areas of emphasis include: (1) building resources, (2) filling gaps in our understanding of biological variation, and (3) systematically characterizing proteome alterations that occur in well-defined disease states, all of which require an organized and collaborative effort.

Oil palm biotechnologies are definitely out of infancy

Although biotechnologies and sustainable development are often considered as antagonists, there is increasing evidence for a role for this approach in the ecological intensification of oil palm culti- vation. Ecological intensification is based on the understanding of how nature functions so as to exploit its resources without destroying it. Living organisms are supported by the genome (DNA) through the action of the transcriptome (RNAs), proteome, metabolome, and ionome, the four basic pillars of func- tional genomics. These pillars represent the sum of all the expressed genes, proteins, metabolites, and elements within an organism. The dynamic response and interaction of these biochemical ''omes'' de- fines how a living system functions, and its study, systems biology, is now one of the biggest challenges in life sciences. In oil palm, as in many major crops, functional genomics is still at its beginning, al- though there are no reasons why oil palm should not rapidly benefit from the fast progresses generated by automated and high-throughput technologies. The success of sequencing projects on model plants has created a widespread interest in exploring the structure and expression patterns of the genome. Indeed, several institutions have now achieved the full sequencing of the oil palm genome, paving the way for the rapid evolution of various genomics-based approaches. Oil palm breeding has provided an average 1% of genetic gain per year since the early 1960s and such an impressive increase in oil yield will be maintained in future generations with a major contribution from biotechnology. Indeed, the re- cent adoption of biotechnological approaches has already proven very useful in major areas such as cloning of outstanding material, identity checking of progenies/mother palms, identification and characterization of genes underlying agricultural traits, etc. Phenotypic differences among individuals are partly the result of quantitative differences in transcript abundance. Using microarray technology, it is now possible to assess the abundance of many transcripts—and, indeed, of the entire known tran- scriptome—simultaneously. Studies which attempt to localize the genetic regulators of gene expression have been carried out in several species, including maize and eucalyptus. Microarrays have been used to determine gene expression levels in segregating populations and identify genomic regions (gene expression QTLs, or eQTLs) explaining transcript variation in co-regulated genes. Our article will focus on recent developments in various plant biotechnology areas in which applications for improving sustainability already exist or will be developed in the near future.

An Integrated Approach to Gene Discovery and Marker Development in Atlantic Cod (Gadus morhua)

Atlantic cod is a species that has been overexploited by the capture fishery. Programs to domesticate this species are underway in several countries, including Canada, to provide an alternative route for production. Selective breeding programs have been successfully applied in the domestication of other species, with genomics-based approaches used to augment conventional methods of animal production in recent years. Genomics tools, such as gene sequences and sets of variable markers, also have the potential to enhance and accelerate selective breeding programs in aquaculture, and to provide better monitoring tools to ensure that wild cod populations are well managed. We describe the generation of significant genomics resources for Atlantic cod through an integrated genomics/selective breeding approach. These include 158,877 expressed sequence tags (ESTs), a set of annotated putative transcripts and several thousand single nucleotide polymorphism markers that were developed from, and have been shown to be highly variable in, fish enrolled in two selective breeding programs. Our EST collection was generated from various tissues and life cycle stages. In some cases, tissues from which libraries were generated were isolated from fish exposed to stressors, including elevated temperature, or antigen stimulation (bacterial and viral) to enrich for transcripts that are involved in these response pathways. The genomics resources described here support the developing aquaculture industry, enabling the application of molecular markers within selective breeding programs. Marker sets should also find widespread application in fisheries management.Electronic supplementary materialThe online version of this article (doi:10.1007/s10126-010-9285-z) contains supplementary material, which is available to authorized users.

Association and Linkage Analysis of Aluminum Tolerance Genes in Maize

BackgroundAluminum (Al) toxicity is a major worldwide constraint to crop productivity on acidic soils. Al becomes soluble at low pH, inhibiting root growth and severely reducing yields. Maize is an important staple food and commodity crop in acidic soil regions, especially in South America and Africa where these soils are very common. Al exclusion and intracellular tolerance have been suggested as two important mechanisms for Al tolerance in maize, but little is known about the underlying genetics.MethodologyAn association panel of 282 diverse maize inbred lines and three F2 linkage populations with approximately 200 individuals each were used to study genetic variation in this complex trait. Al tolerance was measured as net root growth in nutrient solution under Al stress, which exhibited a wide range of variation between lines. Comparative and physiological genomics-based approaches were used to select 21 candidate genes for evaluation by association analysis.ConclusionsSix candidate genes had significant results from association analysis, but only four were confirmed by linkage analysis as putatively contributing to Al tolerance: Zea mays AltSB like (ZmASL), Zea mays aluminum-activated malate transporter2 (ALMT2), S-adenosyl-L-homocysteinase (SAHH), and Malic Enzyme (ME). These four candidate genes are high priority subjects for follow-up biochemical and physiological studies on the mechanisms of Al tolerance in maize. Immediately, elite haplotype-specific molecular markers can be developed for these four genes and used for efficient marker-assisted selection of superior alleles in Al tolerance maize breeding programs.

Antibody-based identification of cell surface antigens: targets for cancer therapy

The identification of novel cell surface antigens present on tumor cells is crucial for developing new cancer therapies. Intact, viable cancer cells, which display cancer-restricted antigens in their native conformation and cellular context, provide a rich source of novel antigens. Antibody-based technologies are being used to probe the surface of intact cancer cells for cancer-specific antigen targets. In addition to identifying new proteins, these approaches are generating monoclonal antibodies (MAbs) to cancer-specific epitopes and nonprotein targets not amenable to genomics-based approaches. The multiple cell-based approaches developing epitope-specific MAbs to cancer antigens is likely to usher in a new era of therapeutic MAb target discovery.

Current Opinions on the Functions of Tocopherol Based on the Genetic Manipulation of Tocopherol Biosynthesis in Plants

As a member of an important group of lipid soluble antioxidants, tocopherols play a paramount role in the daily diet of humans and animals. Recently, genes required for tocochromanol biosynthesis pathway have been identified and cloned with the help of genomics-based approaches and molecular manipulation in the model organisms: Arabidopsis thaliana and Synechocystis sp. PCC 6803. At the basis of these foundations, genetic manipulation of tocochromanol biosynthesis pathway can give rise to strategies that enhance the level of tocochromanol content or convert the constitution of tocochromanol. In addition, genetic manipulations of the tocochromanol biosynthesis pathway provide help for the study of the function of tocopherol in plant systems. The present article summarizes recent advances and pays special attention to the functions of tocopherol in plants. The roles of tocopherol in the network of reactive oxygen species, antioxidants and phytohormones to maintain redox homeostasis and the functions of tocopherol as a signal molecule in chloroplast-to-nucleus signaling to regulate carbohydrate metabolism are also discussed.

Gene expression in the intertidal mussel Mytilus californianus: physiological response to environmental factors on a biogeographic scale

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 356:1-14 (2008) - DOI: https://doi.org/10.3354/meps07354 Gene expression in the intertidal mussel Mytilus californianus: physiological response to environmental factors on a biogeographic scale Sean P. Place, Michael J. O’Donnell, Gretchen E. Hofmann* Department of Ecology, Evolution and Marine Biology, University of California Santa Barbara, Santa Barbara, California 93106-9610, USA *Corresponding author. Email: hofmann@lifesci.ucsb.edu ABSTRACT: We used a cDNA microarray to profile gene expression in the intertidal mussel Mytilus californianus across major portions of its biogeographic range. Overall, the expression pattern for the majority of genes assessed in this study varied significantly as a function of collection site and provides support for the hypothesis that the physiological response to emersion is distinct across populations of M. californianus. Gill tissue was dissected from adult, field-acclimatized M. californianus collected from 4 sites across 17° of latitude along the west coast of North America. First-strand cDNA prepared from 5 biological replicates from each site were competitively hybridized to a 2496 feature cDNA microarray. Gene expression patterns in mussels from Strawberry Hill, Oregon, displayed a unique expression phenotype that was distinct from the other 3 mussel populations sampled. In contrast, mussels sampled from Bamfield, British Columbia, and Jalama Beach, California, showed similar expression patterns. These data suggest that the physiological response of M. californianus to abiotic factors, such as temperature, cannot be predicted by a simple latitudinal gradient. This study highlights the usefulness of genomics-based approaches in assessing physiological responses to environmental variation across large spatial scales. KEY WORDS: Environmental genomics · Mytilus californianus · DNA microarray · Biogeography · Gene expression · Species range · Intertidal invertebrates · Mussel Full text in pdf format NextCite this article as: Place SP, O’Donnell MJ, Hofmann GE (2008) Gene expression in the intertidal mussel Mytilus californianus: physiological response to environmental factors on a biogeographic scale. Mar Ecol Prog Ser 356:1-14. https://doi.org/10.3354/meps07354 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 356. Online publication date: March 18, 2008 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2008 Inter-Research.

Environment Arrays: A Possible Approach for Predicting Changes in Waterborne Bacterial Disease Potential

Current molecular techniques for identifying bacteria in water have proven useful, but they are not reliably predictive of impending disease outbreaks. Genomics-based approaches will help to detect the presence of pathogens quickly and well before they grow into a population that poses a risk to public health. We suggest that genomics is only one component of the toolbox that will be needed to identify emerging waterborne threats. We propose a methodology beyond genomics, based on activity in the mobile genome. This approach makes use of a new device called an environment array. The array will depend upon the same research necessary for genomics-based detection, but will not require an a priori knowledge of virulence genes. Environment arrays are assembled from molecular profiles of the infectious elements that transfer between bacteria. The advantage of the array is that it monitors the activity of the mobile genome, rather than the presence of particular DNA sequences. Environmental arrays should thus be many times more sensitive than traditional hybridization or PCR-based techniques that target already-known DNA sequences. Mobile elements are known to respond to new environmental conditions that may correlate with a chemical contamination or the bloom of bacterial pathogens, potentially allowing for a much broader application in detecting unknown or unanticipated biological and chemical contaminants.

Genomics and marine microbial ecology

Genomics has brought about a revolution in all fields of biology. Before the development of microbial ecology in the 1970s, microbes were not even considered in marine ecological studies. Today we know that half of the total primary production of the planet must be credited to microorganisms. This and other discoveries have changed dramatically the perspective and the focus of marine microbial ecology. The application of genomics-based approaches has provided new challenges and has allowed the discovery of novel functions, an appreciation of the great diversity of microorganisms, and the introduction of controversial ideas regarding the concepts of species, genome, and niche. Nevertheless, thorough knowledge of the traditional disciplines of biology is necessary to explore the possibilities arising from these new insights. This work reviews the different genomic techniques that can be applied to marine microbial ecology, including both sequencing of the complete genomes of microorganisms and metagenomics, which, in turn, can be complemented with the study of mRNAs (transcriptomics) and proteins (proteomics). The example of proteorhodopsin illustrates the type of information that can be gained from these approaches. A genomics perspective constitutes a map that will allow microbiologists to focus their research on potentially more productive aspects.

VITAMIN SYNTHESIS IN PLANTS: Tocopherols and Carotenoids

Carotenoids and tocopherols are the two most abundant groups of lipid-soluble antioxidants in chloroplasts. In addition to their many functional roles in photosynthetic organisms, these compounds are also essential components of animal diets, including humans. During the past decade, a near complete set of genes required for the synthesis of both classes of compounds in photosynthetic tissues has been identified, primarily as a result of molecular genetic and biochemical genomics-based approaches in the model organisms Arabidopsis thaliana and Synechocystis sp. PCC6803. Mutant analysis and transgenic studies in these and other systems have provided important insight into the regulation, activities, integration, and evolution of individual enzymes and are already providing a knowledge base for breeding and transgenic approaches to modify the types and levels of these important compounds in agricultural crops.

Leveraging human genomic information to identify nonhuman primate sequences for expression array development

BackgroundNonhuman primates (NHPs) are essential for biomedical research due to their similarities to humans. The utility of NHPs will be greatly increased by the application of genomics-based approaches such as gene expression profiling. Sequence information from the 3' end of genes is the key resource needed to create oligonucleotide expression arrays.ResultsWe have developed the algorithms and procedures necessary to quickly acquire sequence information from the 3' end of nonhuman primate orthologs of human genes. To accomplish this, we identified terminal exons of over 15,000 human genes by aligning mRNA sequences with genomic sequence. We found the mean length of complete last exons to be approximately 1,400 bp, significantly longer than previous estimates. We designed primers to amplify genomic DNA, which included at least 300 bp of the terminal exon. We cloned and sequenced the PCR products representing over 5,500 Macaca mulatta (rhesus monkey) orthologs of human genes. This sequence information has been used to select probes for rhesus gene expression profiling. We have also tested 10 sets of primers with genomic DNA from Macaca fascicularis (Cynomolgus monkey), Papio hamadryas (Baboon), and Chlorocebus aethiops (African green monkey, vervet). The results indicate that the primers developed for this study will be useful for acquiring sequence from the 3' end of genes for other nonhuman primate species.ConclusionThis study demonstrates that human genomic DNA sequence can be leveraged to obtain sequence from the 3' end of NHP orthologs and that this sequence can then be used to generate NHP oligonucleotide microarrays. Affymetrix and Agilent used sequences obtained with this approach in the design of their rhesus macaque oligonucleotide microarrays.

Functional genomics-based studies of the microbial ecology of hyperthermophilic micro-organisms.

Although much attention has been paid to the genetic, biochemical and physiological aspects of individual hyperthermophiles, how these unique micro-organisms relate to each other and to their natural habitats must be addressed in order to develop a comprehensive understanding of life at high temperatures. Phylogenetic 16 S rRNA-based profiling of samples from various geothermal sites has provided insights into community structure, but this must be complemented with efforts to relate metabolic strategies to biotic and abiotic characteristics in high-temperature habitats. Described here are functional genomics-based approaches, using cDNA microarrays, to gain insight into how ecological features such as biofilm formation, species interaction, and possibly even gene transfer may occur in native environments, as well as to determine what genes or sets of genes may be tied to environmental functionality.

A genomics-based approach to biodefence preparedness.

The anthrax letter attacks in October 2001, followed by the SARS outbreak in early 2003, dramatically illustrated our vulnerability to both deliberate and natural outbreaks of infectious disease. The availability of pathogen genome sequences and high-throughput methods for studying the biology of both pathogens and their hosts have provided new insights into the mechanisms of pathogenesis and host defence. As infectious disease research expands to include major bioterror agents, genomics-based approaches will provide one of the cornerstones of efforts to develop more accurate diagnostics, new therapeutics and vaccines, and further capabilities for microbial forensics.

Human inter-individual DNA sequence variation in candidate genes, drug targets, the importance of haplotypes and pharmacogenomics.

The identification of genes predisposing to human diseases is of paramount importance for understanding the molecular basis of the disease and individually different drug response, and will establish new routes to diagnosis and therapeutic advances of immense medical benefit. A key step common to all strategies for disease gene identification is the systematic analysis of candidate gene sequences to identify specific sequence variations associated with disease or any other phenotype of pharmaceutical relevance. In this article, current concepts and approaches to haplotype-based candidate gene analysis are reviewed. Moreover, a comprehensive summary of recent studies and data on the amount, nature, pattern and structure of genetic variation in candidate genes is given. These data demonstrate altogether remarkable gene sequence and haplotype diversity. Numerous individually different forms of a gene may exist. This presents challenges to the traditional views of the concept of "a" gene with far-reaching implications on the functional analysis of candidate gene variation, on the establishment of "sequence"-"structure"-"function" and complex haplotype/genotype-phenotype relationships, on the identification, evaluation and prioritization of drug targets and the concept of a "personalized medicine" in general. Moreover, present and future approaches to the identification of candidate and disease genes will be addressed. These include whole genome-based approaches such as integrative genomics as well as functional genomics-based approaches to analyze and model complex biological and medical processes. The analysis of whole complex systems in particular will provide the basis to make "maximally informed" guesses on candidate genes and address complex variability patterns in genes as well as complex genotype-phenotype relationships comprehensively at an advanced level.

Cloning and Characterization of Mitochondrial 5-Formyltetrahydrofolate Cycloligase from Higher Plants

5-Formyltetrahydrofolate cycloligase (5-FCL) catalyzes the conversion of 5-formyltetrahydrofolate (5-CHO-H(4)PteGlu(n)) to 5,10-methenyltetrahydrofolate and is considered to be the main means whereby 5-CHO-H(4)PteGlu(n) is metabolized in mammals, yeast, and bacteria. 5-CHO-H(4)PteGlu(n) is known to occur in plants and to be highly abundant in leaf mitochondria. Genomics-based approaches identified Arabidopsis and tomato cDNAs encoding proteins homologous to 5-FCLs of other organisms but containing N-terminal extensions with the features of mitochondrial targeting peptides. These homologs were shown to have 5-FCL activity by characterizing recombinant enzymes produced in Escherichia coli and by functional complementation of a yeast fau1 mutation with the Arabidopsis 5-FCL cDNA. The recombinant Arabidopsis enzyme is active as a monomer, prefers the penta- to the monoglutamyl form of 5-CHO-H(4)PteGlu(n), and has kinetic properties broadly similar to those of 5-FCLs from other organisms. Enzyme assays and immunoblot analyses indicated that 5-FCL is located predominantly if not exclusively in plant mitochondria and that the mature, active enzyme lacks the putative targeting sequence. Serine hydroxymethyltransferase (SHMT) from plant mitochondria was shown to be inhibited by 5-CHO-H(4)PteGlu(n) as are SHMTs from other organisms. Since mitochondrial SHMT is crucial to photorespiration, 5-FCL may help prevent 5-CHO-H(4)PteGlu(n) from reaching levels that would inhibit this process. Consistent with this possibility, 5-FCL activity was far higher in leaf mitochondria than root mitochondria.

Programmed cell death takes flight: genetic and genomic approaches to gene discovery in Drosophila.

Programmed cell death (PCD) is an essential and wide-spread physiological process that results in the elimination of cells. Genes required to carry out this process have been identified, and many of these remain the subjects of intense investigation. Here, we describe PCD, its functions, and some of the consequences when it goes awry. We review PCD in the model system, the fruit fly, Drosophila melanogaster, with a particular emphasis on cell death gene discovery resulting from both genetics and genomics-based approaches.