Location: Cool and Cold Water Aquaculture Research2012 Annual Report
1a. Objectives (from AD-416):
1. Produce a draft reference genome sequence for rainbow trout. 2. Produce a high density SNP chip for rainbow trout.
1b. Approach (from AD-416):
1.a. Sequence the BACs from the physical map minimal tiling path (MTP) using the new Illumina sequencing platform. 1.b. Produce a dense genetic map using 5,000-10,000 SNPs from the Swanson x Whale Rock doubled haploid (DH) recombinant line of Gary Thorgaard to aid in the integration of the genetic and physical maps and the genome sequence assembly. 2.a. Add more SNPs to the current NCCCWA database of 25,000-50,000 putative SNPs using reduced representation sequencing approaches on the Thorgaard’s androgenetic DH lines and additional outbred populations of economic and scientific interest, and based on the genome sequence assembly from objective 1, select and design SNP markers for a chip of up to 50K SNPs. 2.b. Validate a subset of the SNPs using a smaller genotyping assay (e.g. Illumina’s 3K GoldenGate). 2.c. Produce a commercial high-density SNP assay for whole genome simultaneous genotyping in rainbow trout.
3. Progress Report:
This sub-project aims to produce the fish needed to achieve sub-objective 1.b. from the Approach. At Washington State University, we are producing and distributing genetically defined rainbow trout for the project. We produced individuals from eight clonal lines for detection of SNPs differing among the lines using RAD marker analysis. We also will produce at least 440 additional doubled haploid androgenetic progeny from a cross between the Whale Rock XX female and Swanson YY male lines for use in mapping and orientation of the linkage groups. Unfortunately, we did not have enough Whale Rock X Swanson hybrids this past year to produce a large group of doubled haploids, but additional hybrids were produced in 2010 and 2011. The doubled haploid progeny of these hybrids which we produce in 2013 will represent recombination in a male hybrid parent. About fifty of the hybrids produced in 2011 were sex-reversed with beta-estradiol. A gonad from one of these fish was dissected and appeared to be an ovary based on a wet mount (squash) and the observation of oocytes. This suggests that the sex reversal was successful, as the hybrids are otherwise expected to be all male. The doubled haploid progeny of the female hybrids can be used to study recombination in a female hybrid background and thus should have recombination in chromosomal regions lacking in the other cross. We also are rearing about thirty hybrids of the Whale Rock XX female X OSU sex-reversed XX male which were produced in 2010 for use in mapping and orientation of the linkage groups. These hybrids should be all-female and will be used to study recombination in a female hybrid background. They are expected to spawn between January and March 2013. At least 180 doubled haploid progeny will be produced from the female hybrids. They should have recombination in chromosomal regions lacking in the other cross.