A Simultaneous Genetic Screen for Zygotic and Sterile Mutants in a Hermaphroditic Vertebrate (Kryptolebias marmoratus)

Abstract

The mangrove killifish, Kryptolebias marmoratus, is unique among vertebrates due to its self-fertilizing mode of reproduction involving an ovotestis. As a result, it constitutes a simplistic and desirable vertebrate model for developmental genetics as it is easily maintained, reaches sexual maturity in about 100 days, and provides a manageable number of relatively clear embryos. After the establishment and characterization of an initial mutagenesis pilot screen using N-ethyl-N-nitrosourea, a three-generation genetic screen was performed to confirm zygotic mutant allele heritability and simultaneously score for homozygous recessive mutant sterile F2 fish. From a total of 307 F2 fish screened, 10 were found to be 1° males, 16 were sterile, 92 wild-type, and the remaining 189, carriers of zygotic recessive alleles. These carriers produced 25% progeny exhibiting several zygotic phenotypes similar to those previously described in zebrafish and in the aforementioned pilot screen, as expected. Interestingly, new phenotypes such as golden yolk, no trunk, and short tail were observed. The siblings of sterile F2 mutants were used to produce an F3 generation in order to confirm familial sterility. Out of the 284 F3 fish belonging to 10 previously identified sterile families, 12 were found to be 1° males, 69 were wild-type, 83 sterile, and 120 were classified as */+ (either wild-type or carriers) with undefined genotypes. This screen provides proof of principle that K. marmoratus is a powerful vertebrate model for developmental genetics and can be used to identify mutations affecting fertility.

The Genome of the Self-Fertilizing Mangrove Rivulus Fish,Kryptolebias marmoratus: A Model for Studying Phenotypic Plasticity and Adaptations to Extreme Environments

“…A linkage map is now available ( Kanamori et al 2016 ) and N -ethyl N -nitrosourea (ENU) mutagenesis screens have been successful in this species ( Moore et al 2012 ; Sucar et al 2016 ). A transcriptome was recently made available, and shed light on genes that are up- and downregulated during embryonic diapause (the embryo is fully developed but hatching is delayed; [ Mesak et al 2015 ]).…”

Molecular mechanisms of embryonic tail development in the self-fertilizing mangrove killifish Kryptolebias marmoratus

“…1 H,I). These phenotypes appeared with Mendelian ratios consistent with the recessive nature of a mutation occurring in one gene for each line as they originated from different founding F 0 mutated fish ( Sucar et al, 2016 ).

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“…To uncover mechanisms of tail development in the self-fertilizing K. marmoratus , two ENU-mutated lines, R109 and R228 ( Sucar et al, 2016 ), were analysed. R109/shorttail ( stl ) exhibits reduced tail growth with characteristic narrowing at the trunk-tail junction ( Fig.…”

“…This makes the process of mutant screening one generation shorter than other dimorphic animal models and omits the process of identifying families carrying a mutant allele, leading to quicker mutant screens using smaller numbers of fish and tanks. Using the mangrove killifish, Ring's group conducted a pilot screen for zygotic lethal mutants by N-ethyl-N-nitrosourea (ENU)-induced mutagenesis ( Moore et al, 2012 ), followed by a continued F 3 screen to confirm zygotic lethal alleles (old and new) and uncover sterile mutant lines ( Sucar et al, 2016 ). From these lines, we selected two mutants, R109/ shorttail and R228/ balltail , characterized by their unique phenotypes during tail development.…”

Outbreeding depression as a selective force on mixed mating in the mangrove rivulus fish,Kryptolebias marmoratus

“…Eggs were viewed under a stereomicroscope to score each as fertilized, unfertilized, or dead. Fertilized eggs are identifiable by the perivitelline membrane inside the chorion; unfertilized eggs do not have the perivitelline membrane (Harrington 1963, Sucar et al 2016). Dead eggs are identifiable because they are opaque and range in color from white to red.…”