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Combining mutagenesis and genomics in the mouse — closing the phenotype gap

Traditional breeding has been successful in delivering new cultivars with improved grain yield. This is particularly true in the case of maize, with global gains in yield always >2% since the massive introduction of hybrids in the 1960s. The advent of molecular tools in the 1980s, however, demanded an increased knowledge of the whole phenotype for enhancing the breadth of marker-assisted selection. This demand rose during the last decade, and revealed the existence of a large gap between genotypic and phenotypic knowledge, particularly for traits related to the physiological determinants of grain yield (i.e. traits conducive to biomass production and its partitioning). In this chapter, we use maize as a model crop for (1) reviewing general aspects of the physiological model of yield and its dissection in minor traits, (2) examining recent yield gains in major maize-producing countries, and breeding effects on the physiological determinants behind these gains, (3) discussing a field-based approach for phenotyping traits considered critical for yield determination at the crop level, and (4) analyzing possible genetic controls behind the response of yield to its physiological determinants. Opportunities and limitations to the application of field-based phenotyping in maize breeding are also addressed.

In random chemical mutagenesis, gene discovery is driven by phenotypes rather than by hypotheses. A standard dose of N-ethyl-N-nitrosourea results in ∼30 coding mutations in male G1 mice, of which ∼4 can be propagated to homozygosity in 3 generations. In recent years, large-scale screens of such G3 mice for phenotypes of interest to immunologists have revealed clues to the number of genes responsible for key immune responses, such as innate recognition of pathogens and autoantibody production. More than 20 of the phenotypes that exhibit a simple (Mendelian) pattern of inheritance have been mapped. Novel alleles have revealed new pathways of host defense, allergy and autoimmunity.

Significant developments have occurred in our understanding of the mammalian genome thanks to informatics, expression profiling and sequencing of the human and rodent genomes. However, although these facets of genomic analysis are being addressed, analysis of in vivo gene function remains a formidable task. Evaluation of the phenotype of mutants provides powerful access to gene function, and this approach is particularly relevant to the nervous system and behavior. Here, we discuss the complementary mouse genetic approaches of gene-driven, targeted mutagenesis and phenotype-driven, chemical mutagenesis. We highlight an NIH-supported large-scale effort to use phenotype-driven mutagenesis screens to identify mouse mutants with neural and behavioral alterations. Such single-gene mutations can then be used for gene identification using positional candidate gene-cloning methods.

Good osmoregulation is critical to the success of insects, and the Malpighian tubules play a key role in osmoregulation. Recently, the application of genetics and genomics to the Drosophila tubule has revealed far more extensive roles than ion and water transport. Microarray analysis shows that organic solute transporters dominate the tubule transcriptome. The tubule thus has the capability to excrete actively the broadest range of organic solutes and xenobiotics. Such transporters can produce unexpected, emergent roles for the whole tissue; e.g. the tubule is highly resistant to ouabain not because the Na⁺, K⁺ ATPase is unimportant, but because it co-localises with a potent alkaloid excretory mechanism. Reinforcing this role in excretion, the tubule expresses very high levels of a particular cytochrome P450 s, glutathione-S-transferases and alchohol dehydrogenases which suggest that the tubule plays a major role in metabolism and detoxification of both endogenous solutes and xenobiotics, such as insecticides. Additionally, the tubule plays a significant role in immunity; tubules are capable of sensing bacterial challenge, and mounting an effective killing response by secretion of antimicrobial peptides, entirely independent of the fat body, the canonical immune tissue. The tubule has also proved to be a good model for some human renal disease, and to act as an organotypic ‘testbed’ for mammalian genes. The tubule can thus bask in a greatly enhanced reputation as a key tissue for an unexpectedly wide range of functions in the insect.