Forward genetic approach to identify recessive mutations in Ciona. Ciona mutagenesis involves a classical F₂ screen (Moody et al. 1999). The F₀ animals are treated with the chemical mutagen ENU and maintained for several weeks to reduce mosaicism. The mutagenized sperm is then crossed to wild-type animals to obtain heterozygous F₁ progenies. The F₁ animals are individually self-fertilized to produce F₂ progeny that are screened for phenotypic abnormalities. The sperm of mutant lines can then be cryo-preserved for long-term storage. Alternatively, naturally occurring recessive mutations can be isolated by self-crossing single animals from the wild population. The point mutation can be determined by various mapping methods, including linkage analysis of amplified length polymorphism (Jiang et al. 2005).