Summary Site-specific recombinases such as Cre and Flp are invaluable tools for genetic manipulations, but their usage in zebrafish has been limited. Incorporating recently developed flip-excision (FlEx) design that allows stable inversions, we have established zebrafish reporter lines that express bright and ubiquitous EGFP, but switch to express mCherry in the presence of Cre or Flp. Here, we demonstrate the stable inversion in the reporter lines, both in somatic cells and in the germ line by Cre or Flp, and the subsequent reinversion using the other recombinase. Using the reporter lines, we characterized cardiomyocyte-specific Cre lines and neuronal progenitor-specific and tamoxifen-dependent Cre lines. We also used the reporter lines for screening Cre- and Flp-based enhancer trap lines. Similar to the widely used Cre reporter lines in mice, these FlEx-based reporter lines will facilitate the use of recombinases for genetic manipulations in zebrafish.

Conditional mutations are essential for determining the stage-and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre-and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.H igh throughput functional genomic and informatic methods have been developed to interrogate the genome and extract functional predictions about many genes at a time. However, careful phenotypic analysis of genetic mutants remains the sine qua non of reductionist biological science. In most experimental organisms, random mutagenesis is the preferred or only mutagenic technique available. DNA alkylating agents, transposable elements, or retroviruses are traditionally used in these organisms. A major limitation of these traditional genetic methods is that they reveal only the earliest and/or most prominent function of a gene as later functions are masked by the earlier phenotype, which is often lethality. To assess later functions, for example in metabolism, aging, or behavior, conditional alleles are required.The development of conditional alleles has proven a boon to studying gene function in temporally or spatially restricted contexts. Traditional conditional alleles disrupt gene function by changing the environment, for example by increasing the temperature. Engineered conditional alleles disrupt gene function by activating a recombination-mediated molecular switch that ablates gene function in one state, but has no functional consequences in the other state (1, 2...

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Oil produced by castor ( Ricinus communis ) has broad industrial applications. However, knowledge on the genetic diversity, especially genetic alterations that occurred during domestication and subsequent traits selection, of this oil crop is limited. Here, our population genomics analyses show that the Chinese castors have developed a geographic pattern, classified into the southern-, the middle-, and the northern-China groups. We detect a number of candidate genomic loci that are associated with the selection signals during the geographical differentiation and domestication. Using genome-wide association analysis, we identify candidate genes associated with nine agronomically important traits. One of the candidate genes encoding a glycosyltransferase related to cellulose and lignin biosynthesis is associated with both capsule dehiscence and endocarp thickness. We hypothesize that the abundance of cellulose or lignin in endocarp is an important factor for capsule dehiscence. Our results provide foundation for castor breeding and genetic study.