Location: Cool and Cold Water Aquaculture Research
Project Number: 8082-31000-013-02-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Jun 30, 2016
End Date: Dec 31, 2020
Doubled haploid lines have unique value as genomic tools because they have minimal heterozygosity and allow full chromosomal haplotypes to be identified. One of these lines (Swanson) has been used for production of BAC libraries, a BAC fingerprinting physical map and for the rainbow trout genome sequencing project (supported by two previous NRI/AFRI grants). Several have been subjects of important QTL studies. The lines also have important applications in studies of the immune system and in disease research and have been used in a number of such studies. The experimental objectives will include: (1) Establish at least 12 lines at the USDA NCCCWA over a four year period. (2) Transfer at least 100 straws of cryopreserved semen from each line as an ongoing repository. (3) Generate a repository of frozen tissues and genomic DNA from all the lines that will be available for the research community. (4) Conduct baseline karyotype analysis (including presence or absence of chromosome fragments) and SNP typing (including re-sequencing) of all the lines. (5) Attempt to induce sex reversal to females in the YY lines and test the fertility of the resulting females. This will provide additional options for propagation and crossing of lines. (6) Re-evaluate the use of hydrostatic pressure for blockage of the first cleavage in propagation of the lines. This may result in improved survival and larger sample size for genetic studies.
Our approaches for these experimental objectives will be: (1) Establish the lines at NCCCWA by inducing androgenesis or gynogenesis and transferring certified disease-free eyed eggs or juvenile fish. Paul Wheeler, hatchery manager at WSU, would also travel to NCCCWA to guide and train personnel in how to apply androgenesis and gynogenesis. (2) Apply established sperm cryopreservation methods to develop semen repositories at the NCCCWA and at least one other site. (3) Prepare chromosomes from embryos and type SNPs using established chips to characterize the lines and assure line integrity. (4) Expose fry to 17-beta estradiol to induce male to female sex reversal in YY and XY individuals. (5) Apply hydrostatic pressure at different times after fertilization to optimize suppression of first cleavage.