Genetic and genomic approaches to study placental development

Recent technological advances in genetic manipulation and expression profiling offer excellent opportunities to elucidate the molecular mechanisms controlling developmental processes during embryogenesis. Thus, this revolution also strongly benefits studies of the molecular genetics of placental development. Here we review the findings of several expression profiling analyses in extraembryonic tissues and assess how this work can contribute to the identification of essential components governing placental development. We further discuss the relevance of these components in the context of genetic manipulation experiments. In conclusion, the intelligent combination of genetic and genomic approaches will substantially accelerate the progress in identifying the key molecular pathways of placental development.     

Genetic and genomic approaches to develop rice germplasm for problem soils

Soils that contain toxic amounts of minerals or are deficient in essential plant nutrients are widespread globally and seriously constrain rice production. New methods are necessary to incorporate the complex adaptive traits associated with tolerance of these abiotic stresses, while simultaneously retaining the high yield potential of rice varieties when conditions are favorable. Significant progress in the genetic characterization of stress response pathways and recent advances in genomics have provided powerful tools for in-depth dissection of tolerance mechanisms. Additionally, tolerance of most of these abiotic stresses in rice is controlled by a few QTLs with large effects despite the intricacy of the numerous traits involved. Genetic dissection of these QTLs and their incorporation into high-yielding varieties will significantly enhance and stabilize rice productivity in these problem soils. Current efforts at IRRI and in rice breeding programs worldwide are seeking to explore diverse germplasm collections and genetically dissect the causal mechanisms of tolerance to facilitate their use in breeding. This review focuses on salinity and P and Zn deficiency as the major problems encountered in rice soils, and examines current understanding of the mechanisms involved and efforts toward germplasm improvement.     

Genetic approaches to disease and regeneration

Cardiovascular disease is largely a consequence of coronary artery blockage through excessive proliferation of smooth muscle cells. It in turn leads to myocardial infarction and permanent and functionally devastating tissue damage to the heart wall. Our studies have revealed that elastin is a primary player in maintaining vascular smooth muscle cells in their dormant state and thus may be a useful therapeutic in vascular disease. By studying zebrafish, which unlike humans, can repair damage to heart muscle, we have begun to uncover some of the genes that seem necessary to undertake the de-differentiation steps that currently fail and prevent the formation of new proliferating cardiomyocytes at the site of damage in a mammalian heart.     

Comparisons of single-stage and two-stage approaches to genomic selection

Genomic selection (GS) is a method for predicting breeding values of plants or animals using many molecular markers that is commonly implemented in two stages. In plant breeding the first stage usually involves computation of adjusted means for genotypes which are then used to predict genomic breeding values in the second stage. We compared two classical stage-wise approaches, which either ignore or approximate correlations among the means by a diagonal matrix, and a new method, to a single-stage analysis for GS using ridge regression best linear unbiased prediction (RR-BLUP). The new stage-wise method rotates (orthogonalizes) the adjusted means from the first stage before submitting them to the second stage. This makes the errors approximately independently and identically normally distributed, which is a prerequisite for many procedures that are potentially useful for GS such as machine learning methods (e.g. boosting) and regularized regression methods (e.g. lasso). This is illustrated in this paper using componentwise boosting. The componentwise boosting method minimizes squared error loss using least squares and iteratively and automatically selects markers that are most predictive of genomic breeding values. Results are compared with those of RR-BLUP using fivefold cross-validation. The new stage-wise approach with rotated means was slightly more similar to the single-stage analysis than the classical two-stage approaches based on non-rotated means for two unbalanced datasets. This suggests that rotation is a worthwhile pre-processing step in GS for the two-stage approaches for unbalanced datasets. Moreover, the predictive accuracy of stage-wise RR-BLUP was higher (5.0–6.1 %) than that of componentwise boosting.     

Genetic approaches to auxin action

Answers to long-standing questions concerning the molecular mechanism of auxin action and auxin's exact functions in plant growth and development are beginning to be uncovered through studies using mutant and transgenic plants. We review recent work in this area in vascular plants. A number of conclusions can be drawn from these studies. First, auxin appears essential for cell division and viability, as auxin auxotrophs isolated in tissue culture are dependent on auxin for growth and cannot be regenerated into plants even when auxin is supplied exogenously. Secondly, plants with transgenes that alter auxin levels are able to regulate cellular auxin concentrations by synthesis and conjugation; wild-type plants are probably also capable of such regulation. Thirdly, the phenotypes of transgenic plants with altered auxin levels and of mutant plants with altered sensitivity to auxin confirm earlier physiological studies which indicated a role for auxin in regulation of apical dominance, in development of roots and vascular tissue, and in the gravitropic response. Finally, the cloning of a mutationally identified gene important for auxin action, along with accumulating biochemical evidence, hints at a major role for protein degradation in the auxin response pathway.     

Genetic approaches to common diseases

Combined molecular and epidemiological studies are advancing our understanding of the genetic basis of multifactorial diseases. Several of the results obtained during the past year highlight methodological issues associated with these approaches. For example, the affected sib-pair method has been applied successfully to detect linkage between the angiotensinogen gene and susceptibility to hypertension, and a large multi-centre epidemiological study has demonstrated association of a polymorphism of the angiotensin-converting enzyme gene with increased risk of myocardial infarction. The study of Mendelian forms of multifactorial diseases has also led to many new results. These include the characterization of mutations in the glucokinase gene in maturity onset diabetes of the young, localization to chromosome 2 of a gene involved in familial colon cancer, and localization to chromosome 19 of a gene responsible for hemiplegic migraine. New insights have been provided into the genetics of multifactorial disorders such as diabetes and hypertension through the study of animal models. Localization of susceptibility loci in such models has recently led to the identification of new candidate genes that may be implicated in disease.     

Genetic approaches to auxin action

Answers to long-standing questions concerning the molecular mechanism of auxin action and auxin's exact functions in plant growth and development are beginning to be uncovered through studies using mutant and transgenic plants. We review recent work in this area in vascular plants. A number of conclusions can be drawn from these studies. First, auxin appears essential for cell division and viability, as auxin auxotrophs isolated in tissue culture are dependent on auxin for growth and cannot be regenerated into plants even when auxin is supplied exogenously. Secondly, plants with transgenes that alter auxin levels are able to regulate cellular auxin concentrations by synthesis and conjugation; wild-type plants are probably also capable of such regulation. Thirdly, the phenotypes of transgenic plants with altered auxin levels and of mutant plants with altered sensitivity to auxin confirm earlier physiological studies which indicated a role for auxin in regulation of apical dominance, in development of roots and vascular tissue, and in the gravitropic response. Finally, the cloning of a mutationally identified gene important for auxin action, along with accumulating biochemical evidence, hints at a major role for protein degradation in the auxin response pathway.     

Genetic and genomic approaches for R-gene mediated disease resistance in tomato: retrospects and prospects

Tomato (Solanum lycopersicum) is one of the world's most important vegetable crops. Managing the health of this crop can be particularly challenging; crop resistance may be overcome by new pathogen races while new pathogens have been introduced by global agricultural markets. Tomato is extensively used as a model plant for resistance studies and much has been attained through both genetic and biotechnological approaches. In this paper, we illustrate genomic methods currently employed to preserve resistant germplasm and to facilitate the study and transfer of resistance genes, and we describe the genomic organization of R-genes. Patterns of gene activation during disease resistance response, identified through functional approaches, are depicted. We also describe the opportunities offered by the use of new genomic technologies, including high-throughput DNA sequencing, large-scale expression data production and the comparative hybridization technique, whilst reporting multifaceted approaches to achieve genetic tomato disease control. Future strategies combining the huge amount of genomic and genetic data will be able to accelerate development of novel resistance varieties sustainably on a worldwide basis. Such strategies are discussed in the context of the latest insights obtained in this field.     

Genetic approaches to harvesting lichen products

Lichens are symbiotic associations between fungi, green algae and/or cyanobacteria. They have a varied chemistry and produce many polyketide-derived compounds, including some, such as depsides and depsidones, that are rarely reported elsewhere. Although lichens have been appreciated in traditional medicines, their value has largely been ignored by the modern pharmaceutical industry because difficulties in establishing axenic cultures and conditions for rapid growth preclude their routine use in most conventional screening processes. Recently, molecular genetic techniques using PCR, genomic library construction and heterologous expression have provided an alternative approach to begin exploring the diversity of polyketide biosynthetic pathways in lichens. The techniques can be expanded to cover other pathway types and be integrated with conventional culture collection-based screening to provide a comprehensive search for novel chemical entities in these organisms.     

Genetic approaches to memory storage.

The ability to remember is perhaps the most significant and distinctive feature of our mental life. We are who we are largely because of what we have learned and what we remember. In turn, impairments in learning and memory can lead to disorders that range from the moderately inconvenient benign senescent forgetfulness associated with normal aging to the devastating memory losses associated with Alzheimer disease. Of the various, higher-cognitive abilities that human beings possess, such as reasoning and language, memory is the only one that can be studied effectively in simple experimental organisms that are accessible to genetic manipulation, such as snails, flies and mice. In these organisms, the effectiveness of genetic approaches in the study of memory has improved significantly in the past five years. Below we review these advances.     

Genetic approaches to studying peroxisome biogenesis

How proteins are imported into peroxisomes is a question attracting considerable interest at present. Peroxisomal proteins, including the integral membrane proteins of the membrane bounding the peroxisome, are synthesized on free cytoplasmic ribosomes. They assemble post-translationally into pre-existing peroxisomes. New peroxisomes are believed to form exclusively by division of old ones. Few molecular details of this process have been elucidated so far, but genetic approaches are now beginning to identify the proteins catalysing peroxisome assembly.     

Genetic approaches to understanding sugar-response pathways

Plants as photoautotrophic organisms are able to produce the carbohydrates they require and have developed mechanisms to co-ordinate carbohydrate production and its metabolism. Carbohydrate-derived signals regulate the expression of genes involved in both photosynthesis and metabolism, and control carbohydrate partitioning. A number of genetic approaches have been initiated to understand sugar-response pathways in plants and identify the components involved. Screening strategies to date have been based on the effects of high sugar media on early seedling development or on changes in the enzyme activity or expression of sugar-responsive genes. These screens have established roles for plant hormones in sugar-response pathways, in particular for abscisic acid. The present emphasis on the role of plant hormones in sugar responses is due to the fact that mutants could be readily identified as belonging to these established pathways, but also results from the nature of the mutant screens in use. Progress is being made on the identification of mutants and genes that may be specific to sugar-signalling pathways. It is also expected that the modification of existing screens may target sugar-signalling pathways more directly. Genetic approaches may be especially useful in identifying components of novel signalling pathways unique to plants, and their combination with genomic and molecular approaches will guide future research.     

Adapting legume crops to climate change using genomic approaches

Our agricultural system and hence food security is threatened by combination of events, such as increasing population, the impacts of climate change, and the need to a more sustainable development. Evolutionary adaptation may help some species to overcome environmental changes through new selection pressures driven by climate change. However, success of evolutionary adaptation is dependent on various factors, one of which is the extent of genetic variation available within species. Genomic approaches provide an exceptional opportunity to identify genetic variation that can be employed in crop improvement programs. In this review, we illustrate some of the routinely used genomics‐based methods as well as recent breakthroughs, which facilitate assessment of genetic variation and discovery of adaptive genes in legumes. Although additional information is needed, the current utility of selection tools indicate a robust ability to utilize existing variation among legumes to address the challenges of climate uncertainty.     

Genetic approaches to study Legionella pneumophila pathogenicity

Abstract: Legionella pneumophila is an intracellular pathogen replicating in human macrophages during the course of infection of the lungs, infection by legionellae often leads to severe pneumonia, termed Legionnaires' disease. Genetic approaches to identify the factors responsible for L. pneumophila pathogenicity started with the construction of genomic libraries in Escherichia coli. Various L. pneumophila -specific genes were cloned in E. coli K-12 by identifaction using functional assays, antibody screening and hybridization ('reverse genetics'). By disrupting the genes via allelic exchange, mutants have been created to assess the influence of the factors on pathogenicity. Among the cloned genes, only for the gene product of the mip gene, encoding a 24-kDa surface-associated protein (macrophage infectivity potentiator) unequivocal evidence for its contribution to pathogenicity could be provided. Two hemolytic factors that have been cloned do not seem to play a role in L. pneumophila pathogenicity. Genetic systems for transposon mutagenesis of the L. pneumophila genome (Tn5, Tn903dlIlacZ, MudphoA), including TnphoA shuttle mutagenesis, have been established and specifically adapted to identify mutants which displayed an impaired capability to multiply inside macrophages and with a reduced in vivo virulence. Furthermore, by complementation of avirulent mutants, genetic loci could be identified which restored the virulence.