Analysis of expressed sequence tags from Actinidia : Applications of a cross species EST database for gene discovery in the areas of flavor, health, color and ripening

Background Kiwifruit (Actinidia spp.) are a relatively new, but economically important crop grown in many different parts of the world. Commercial success is driven by the development of new cultivars with novel consumer traits including flavor, appearance, healthful components and convenience. To increase our understanding of the genetic diversity and gene-based control of these key traits in Actinidia, we have produced a collection of 132,577 expressed sequence tags (ESTs). Results The ESTs were derived mainly from four Actinidia species (A. chinensis, A. deliciosa, A. arguta and A. eriantha) and fell into 41,858 non redundant clusters (18,070 tentative consensus sequences and 23,788 EST singletons). Analysis of flavor and fragrance-related gene families (acyltransferases and carboxylesterases) and pathways (terpenoid biosynthesis) is presented in comparison with a chemical analysis of the compounds present in Actinidia including esters, acids, alcohols and terpenes. ESTs are identified for most genes in color pathways controlling chlorophyll degradation and carotenoid biosynthesis. In the health area, data are presented on the ESTs involved in ascorbic acid and quinic acid biosynthesis showing not only that genes for many of the steps in these pathways are represented in the database, but that genes encoding some critical steps are absent. In the convenience area, genes related to different stages of fruit softening are identified. Conclusion This large EST resource will allow researchers to undertake the tremendous challenge of understanding the molecular basis of genetic diversity in the Actinidia genus as well as provide an EST resource for comparative fruit genomics. The various bioinformatics analyses we have undertaken demonstrates the extent of coverage of ESTs for genes encoding different biochemical pathways in Actinidia.

Next-generation sequencing: A frameshift in skeletal dysplasia gene discovery

In the last decade, huge breakthroughs in genetics - driven by new technology and different statistical approaches - have resulted in a plethora of new disease genes identified for both common and rare diseases. Massive parallel sequencing, commonly known as next-generation sequencing, is the latest advance in genetics, and has already facilitated the discovery of the molecular cause of many monogenic disorders. This article describes this new technology and reviews how this approach has been used successfully in patients with skeletal dysplasias. Moreover, this article illustrates how the study of rare diseases can inform understanding and therapeutic developments for common diseases such as osteoporosis. © International Osteoporosis Foundation and National Osteoporosis Foundation 2013.

New approaches to gene discovery with animal models of obesity and diabetes

DNA-based approaches to the discovery of genes contributing to the development of type 2 diabetes have not been very successful despite substantial investments of time and money. The multiple gene-gene and gene-environment interactions that influence the development of type 2 diabetes mean that DNA approaches are not the ideal tool for defining the etiology of this complex disease. Gene expression-based technologies may prove to be a more rewarding strategy to identify diabetes candidate genes. There are a number of RNA-based technologies available to identify genes that are differentially expressed in various tissues in type 2 diabetes. These include differential display polymerase chain reaction (ddPCR), suppression subtractive hybridization (SSH), and cDNA microarrays. The power of new technologies to detect differential gene expression is ideally suited to studies utilizing appropriate animal models of human disease. We have shown that the gene expression approach, in combination with an excellent animal model such as the Israeli sand rat (Psammomys obesus), can provide novel genes and pathways that may be important in the disease process and provide novel therapeutic approaches. This paper will describe a new gene discovery, beacon, a novel gene linked with energy intake. As the functional characterization of novel genes discovered in our laboratory using this approach continues, it is anticipated that we will soon be able to compile a definitive list of genes that are important in the development of obesity and type 2 diabetes.

Obesity and diabetes gene discovery approaches

New treatments are currently required for the common metabolic diseases obesity and type 2 diabetes. The identification of physiological and  biochemical factors that underlie the metabolic disturbances observed in obesity and type 2 diabetes is a key step in developing better therapeutic outcomes. The discovery of new genes and pathways involved in the  pathogenesis of these diseases is critical to this process, however  identification of genes that contribute to the risk of developing these diseases represents a significant challenge as obesity and type 2 diabetes are complex diseases with many genetic and environmental causes. A number of diverse approaches have been used to discover and validate potential new targets for obesity and diabetes. To date, DNA-based approaches using candidate gene and genome-wide linkage analysis have had limited success in identifying genomic regions or genes involved in the development of these diseases. Recent advances in the ability to evaluate linkage analysis data from large family pedigrees using variance components based linkage analysis show great promise in robustly identifying genomic regions associated with the development of obesity and diabetes. RNA-based technologies such as cDNA microarrays have identified many genes differentially expressed in tissues of healthy and diseased subjects. Using a combined approach, we are endeavouring to focus attention on differentially expressed genes located in chromosomal regions previously linked with obesity and / or diabetes. Using this strategy, we have identified Beacon as a potential new target for obesity and diabetes.

Novel gene discovery in the muscle of obese-diabetic Psammomys obesus

Metabolism in Psammomys obesis, a polygenic animal model of obesity and type 2 diabetes is associated with dysregulated nocturnal fat oxidation in diabetic animals. Furthermore, a new gene called AGT-203 has been identified. Evidence indicates that AGT-203 is involved in abnormal glucose metabolism leading to the proposition that AGT-203 is a new candidate gene for type 2 diabetes.

Chicken skeletal muscle-associated macroarray for gene discovery

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Gene discovery in Boophilus microplus, the cattle tick - the transcriptomes of ovaries, salivary glands, and hemocytes

The quest for new control strategies for ticks can profit from high throughput genomics. In order to identify genes that are involved in oogenesis and development, in defense, and in hematophagy, the transcriptomes of ovaries, hemocytes, and salivary glands from rapidly ingurgitating females, and of salivary glands from males of Boophilus microplus were PCR amplified, and the expressed sequence tags (EST) of random clones were mass sequenced. So far, more than 1,344 EST have been generated for these tissues, with approximately 30% novelty, depending on the the tissue studied. To date approximately 760 nucleotide sequences from B. microplus are deposited in the NCBI database. Mass sequencing of partial cDNAs of parasite genes can build up this scant database and rapidly generate a large quantity of useful information about potential targets for immunobiological or chemical control.

Gene Discovery in Nonsyndromic Cleft Lip with or without Cleft Palate

Nonsyndromic cleft lip with or without cleft palate (NSCLP), a common, complex orofacial birth defect that affects approximately 4,000 newborns each year in the United States, is caused by both genetic and environmental factors. Orofacial clefts affect the mouth and nose, causing severe deformity of the face, which require medical, dental and speech therapies. Despite having substantial genetic liability, less than 25% of the genetic contribute to NSCLP has been identified. The studies described in this thesis were performed to identify genes that contribute to NSCLP and to demonstrate the role of these genes in normal craniofacial development. Using genome scan and candidate gene approaches, novel associations with NSCLP were identified. These include MYH9 (7 SNPs, 0.009≤p<0.05), Wnt3A (4 SNPs, 0.001≤p≤0.005), Wnt11 (2 SNPs, 0.001≤p≤0.01) and CRISPLD2 (4 SNPs, 0.001≤p<0.05). The most interesting findings were for CRISPLD2. This gene is expressed in the fused mouse palate at E17.5. In zebrafish, crispld2 localized to the craniofacial region by one day post fertilization. Morpholino knockdown of crispld2 resulted in a lower survival rates and altered neural crest cell (NCC) clustering. Because NCCs form the tissues that populate the craniofacies, this NCC abnormality resulted in cartilage abnormalities of the jaw including fewer ceratobranchial cartilages forming the lower jaw (three pairs compared to five) and broader craniofacies compared to wild-type zebrafish. These findings suggest that the CRISPLD2 gene plays an important role in normal craniofacial development and perturbation of this gene in humans contributes to orofacial clefting. Overall, these results are important because they contribute to our understanding of normal craniofacial development and orofacial clefting etiology, information that can be used to develop better methods to diagnose, counsel and potentially treat NSCLP patients.

Gene discovery in the wood-forming tissues of poplar: Analysis of 5,692 expressed sequence tags

A rapidly growing area of genome research is the generation of expressed sequence tags (ESTs) in which large numbers of randomly selected cDNA clones are partially sequenced. The collection of ESTs reflects the level and complexity of gene expression in the sampled tissue. To date, the majority of plant ESTs are from nonwoody plants such as Arabidopsis, Brassica, maize, and rice. Here, we present a large-scale production of ESTs from the wood-forming tissues of two poplars, Populus tremula L. × tremuloides Michx. and Populus trichocarpa ‘Trichobel.’ The 5,692 ESTs analyzed represented a total of 3,719 unique transcripts for the two cDNA libraries. Putative functions could be assigned to 2,245 of these transcripts that corresponded to 820 protein functions. Of specific interest to forest biotechnology are the 4% of ESTs involved in various processes of cell wall formation, such as lignin and cellulose synthesis, 5% similar to developmental regulators and members of known signal transduction pathways, and 2% involved in hormone biosynthesis. An additional 12% of the ESTs showed no significant similarity to any other DNA or protein sequences in existing databases. The absence of these sequences from public databases may indicate a specific role for these proteins in wood formation. The cDNA libraries and the accompanying database are valuable resources for forest research directed toward understanding the genetic control of wood formation and future endeavors to modify wood and fiber properties for industrial use.

Gene discovery and gene function assignment in filamentous fungi

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.