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.
A minimal tilling path (MTP) of approximately 15,000 BAC clones was extracted from the rainbow trout physical map and the MTP clones were picked from the original BAC libraries and re-arrayed into 96-well plates. DNA was extracted from each of the MTP clones and pooled in an indexed approach for tagging and sequencing with the Illumina sequencing platform. Three mate-pairs libraries were prepared from Swanson genomic DNA and sequenced to aid in assembling sequence scaffolds for the BACs using sequence assembly software programs. The assembly of sequence scaffolds is currently underway to enable producing a reference genome sequence assembly that will be guided by the rainbow trout physical map. In addition, a high density genetic map composed of approximately 5,000 SNPs and derived from the Swanson doubled haploid line was produced to aid in the ordering and assembly of the sequence scaffolds into chromosomes. We are also conducting a comprehensive SNP markers discovery and validation effort to characterize their polymorphism between and within rainbow trout populations using next-generation sequencing of Restriction-site Associated DNA (RAD) tags. Tissue samples were collected from 24 rainbow trout populations that are of economic and scientific interest and DNA is currently being extracted from those samples.