1a. Objectives (from AD-416)
Specific Aim 1. Use broad species diversity to identify and select new SNPs that are uniformly distributed across the turkey genome; Specific Aim 2. Validate and characterize SNPs in relevant commercial populations.
1b. Approach (from AD-416)
For the emerging turkey genome sequence to successfully be applied to gene discovery, there is a need to improve the process of SNP discovery and create high-density SNP genotyping assays. Advances in DNA sequencing technology have increased the capacity to the extent that the reduced representation library model is not the only approach for SNP discovery. Furthermore, aligning contigs containing putative SNPs against a reference genome sequence will enable the detection of contigs that map to a single, unique region of the genome, which increases the chance of obtaining a reliable genotype five times. We hypothesize that deep, shotgun sequencing (24-30X) of turkeys from a broad pedigree range (commercial, historical, heritage and ancestral populations) can be used to rapidly identify and characterize SNPs for creating a high-density (60K) SNP genotyping platform for use by the commercial industry and research communities. This Tools and Resources proposal represents an innovative, integrated approach to rapidly and cost effectively identify and validate SNPs and incorporates second generation sequencing technology to ensure broad genome coverage and the depth to uncover highly probable SNPs, clusters of segregating SNPs based on lines as well as SNPs lost through domestication.
3. Progress Report
The goal of this project is to provide the content for development of a single nucleotide polymorphism (SNP) chip for use by the commercial turkey industry to interrogate genetic differences (single nucleotide variations in the genome’s DNA) and potentially enable the selection for preferred traits or genetic merit in turkey, particularly reproductive traits with low heritibility. We expect to generate a minimum of 500,000 SNPs that are uniformly distributed across the turkey genome, using the recently sequenced turkey genome (version 2.01) as the reference genome. Blood or tissue samples were obtained from male turkeys representing 11 relevant lines (n=3 males/line) for DNA extraction and cDNA library construction. Seven of the lines were elite pedigree lines provided by 2 turkey primary breeder companies. Two heritage breeds were sampled, the Narragansett and Royal Palm, as well as the Beltsville Small White, a specialty breed. Historic tissue samples (circa 1899) were obtained from the South Mexican turkey, believed to be the wild ancestral species of the modern commercial bird. The genomic DNA of all birds was sequenced individually using the Illumina Genome Analyzer II (120 bp read length; PE; 6X coverage per bird). The average individual sequence depth, based on the turkey’s genome size of 1 billion bases, was 4.3X with 12.8X coverage per line/breed. To date, 4 million SNPs have been discovered from 3 elite commercial, 1 heritage and the Beltville Small White lines. The total number of SNPs and number of segregated SNPs for each of the 5 lines was similar (1,751,036 SNP and 1,012,966, respectively); however, the Beltsville Small White had a higher number of fixed SNP (1,049,800) than the other 4 lines (mean SNP = 660,135). Individual male Beltsville Small White had higher numbers of homozygous SNP (1,053,216) than males from the other 4 lines (range, 583,191-795,330). The number of heterozygous SNP of individual males from all lines ranged from 206,285 to 579,232.