TURKEY GENOMIC RESEARCH AND GENETIC IMPROVEMENT: DEVELOPMENT OF HIGH-DENSITY SNP CONTENT FOR DESIGN OF A GENOME-WIDE ASSAY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0220757
• Grant No.: 2010-65205-20428
• Project No.: MDR-2009-03318
• Proposal No.: 2009-03318
• Program Code: 92120
• Project Start Date: Feb 1, 2010
• Project End Date: Jan 31, 2014
• Grant Year: 2010

Project Director
Long, J. A.

Recipient Organization
AGRICULTURAL RESEARCH SERVICE
RM 331, BLDG 003, BARC-W
BELTSVILLE,MD 20705-2351

Performing Department
(N/A)

Non Technical Summary
The 3.7 billion dollar US commercial turkey industry has achieved dramatic genetic improvements in growth rate and muscle conformation since the introduction of the broad-breasted white variety in the 1950's. The average market ages and weights of commercial turkeys have become inversely proportional over time, where modern birds develop to desired market weight at a much lower age than the founder flocks. This genetic improvement is attributed largely to selective breeding programs that rely on highly heritable phenotypic traits, such as body size and breast muscle development. Virtually all the genetic selection work is accomplished with pure lines maintained by primary breeders. The numbers of pure-line birds are relatively small compared to the numbers that are eventually produced after 3 to 4 generations of hybridization and multiplication through grandparent and parent lines to commercial stocks. Although significant advances have been made with traditional selection/breeding programs, there are important economic traits that are either expensive/difficult to measure (e.g. disease resistance) or exhibit low heritability (e.g. reproduction). It is increasingly clear that future genetic improvements in the turkey industry will rely more on genotypic characterization rather than phenotypic expression of desired traits. The goal of this project is to provide the content for development of a SNP chip for use by the commercial turkey industry similar to the highly successful Illumina BovineSNP50 SNP chip. The Illumina GAII will be used to identify genome-wide SNP using targeted deep sequence from several DNA pools. DNA pools will be selected considering relevance to commercial lineages, representation of unique research populations, and representation of broad species diversity (e.g., pedigree lines; Beltsville Small White; heritage breeds; ancestral breeds). We expect to generate a minimum of 500,000 SNPs that are uniformly distributed across the turkey genome with high MAF (>0.05). The constructed libraries and sequencing strategy are estimated to allow the selection of at least 1 million SNPs for future incorporation into a high-density genotyping platform. Genome wide marker assisted selection has the potential to improve the accuracy of breeding values and genetic progress for traits where such accuracy is currently relatively low. This is particularly relevant for reproductive, health and quality traits, where evaluating genetic potential through testing of offspring or sibs is very ineffective and expensive for the turkey industry. Our goal is to provide the tools that the turkey industry needs for commercially viable genetic prediction and that the research community will need to explore the structure and function of the turkey genome.

Animal Health Component

(N/A)

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
303	3230	1040	25%
303	3230	1080	25%
304	3230	1040	25%
304	3230	1080	25%

Knowledge Area
303 - Genetic Improvement of Animals; 304 - Animal Genome;

Subject Of Investigation
3230 - Turkey, live animal;

Field Of Science
1080 - Genetics; 1040 - Molecular biology;

Keywords

turkey

snp discovery

genome

beltsville small white turkey

heritage breeds

genotyping

marker assisted selection

Goals / Objectives
Historically, quantitative genetics-based selection has been the primary strategy of genetic improvement in the commercial turkey. The availability of genome-based selection has the power to transform the breeder operation and incorporate previously unavailable genetic information into commercial lines. Single nucleotide polymorphisms represent the most abundant known source of genetic variation within the genome and are linked to heritable inter-individual differences in complex phenotypes. However, biological processes underlying complex traits are themselves often very complicated and require the assessment of multiple loci jointly to be more informative and error proof than individual markers. To date about 11,000 SNPs have been identified in the turkey and, if the SNP frequency in turkey mimics that of the chicken, this represents only about 0.004%. This points to the need for in-depth SNP discovery across the genome to eventually address, through genetic association studies, factors that regulate the establishment of desirable reproductive and production traits. The primary goal of this proposal is SNP discovery and validation to generate a platform for the subsequent construction of a high-density (60K) SNP chip, utilizing current and newly characterized SNPs, to: (1) significantly increase the number of validated and annotated SNPs in the public database and (2) define relatedness among various domestic and non-domestic lines. The project is designed to complement the de novo whole genome shotgun sequencing effort of the International Turkey Genome Sequencing Consortium. Together, these complimentary research efforts will deliver to industry partners the knowledge and a robust tool to facilitate the immediate application of genome-wide selection for the prediction of genetic merit within turkey breeding programs.

Project Methods
During Phase 1 of the project, we will generate DNA libraries from the genomic DNA of 24 birds representing 8 relevant turkey lines and breeds. Individual genomic libraries will be constructed from 3 representative birds from the following: 1)Pure line A; 2)Pure line B; 3)Pure line C; 4)Pure line D; 5)Beltsville Small White; 6)Narragansett heritage breed; 7)Royal Palm heritage breed and 8) non-domestic wild turkey (from 3 geographic locations: NY, MI, VA). A total of 48 haploid genomes will be represented. Following clustering and amplification, we will compile sequence data from 2 flow cells per bird using the Illumina Genome Analyzer. Our ongoing turkey genome sequencing efforts with single reads of 80 bp and 8-10 x 106 reads per flow cell lane yields 4 Gb per flow cell, and we fully expect this yield to increase to provide ~10-12 Gb per run, or 1.25-1.5 Gb per lane using the recently upgraded GA II and version 3 kits. Nonetheless, with a more conservative estimate of 4 GB per flow cell, we expect to generate a minimum sequence depth of 8X per bird or 24X per line. llumina GA sequencers now can generate an expected 4-7 Gb per run. The anticipated number of sequences produced within the project will be: (3 birds) x (8X depth) x (8 lines) x (1.2 Gb genome size) = 230.4 Gb. Sequences obtained will be aligned to generate contigs that can be aligned with our draft turkey genome or, where gaps are present in the scaffolding, the chicken genome. Using this approach we will obtain the information for a pool of 16 haplotypes for across the 8 lines and a total of 48 haplotypes in the total data set. This will allow accurate estimations of the overall MAF for the SNPs identified. Furthermore, we will be able to identify SNPs that have relatively high MAF in each of the individual lines. During Phase 2 of the project, we will select representative SNPs to be validated before scaling up the genotype assay. From this group, 200 SNPs will be selected that represent the total number of SNPs identified and that will allow the verification of the different thresholds used. These 200 SNPs will be genotyped in the 24 birds that were used to identify the SNPs under Phase 1. Genotyping will be done using Illumina's BeadXpress system. Secondly, we will select a set of 60,000 SNPs from the total 1 million SNPs identified in Phase 1 based on the following criteria: (1)MAF of the SNP. The aim is to select SNPs with high MAF to optimize the information content of the final SNP chip. (2)Position of the SNP on the turkey genome using the turkey draft genome sequence currently under construction. The chicken genome sequence and the chicken-turkey comparative map will provide the means of predicting the location of the SNPs on the turkey genome. (3)Type II Infinium assay. This assay requires only a single probe on the Beadchip and therefore will increase the number of SNPs to be added to the chip. (4)Illumina design score of at least 0.7 to optimize the conversion rate of the SNP in the assay. (5)Whether SNPs have already been validated and used in independent genotyping assays. (6)SNPs that are segregating in the majority of the commercial turkey breeds.

Progress 02/01/10 to 01/31/14

Outputs
Target Audience:Target audiences included primary turkey breeding companies and turkey researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Results have been disseminated by two peer-reviewed publications, as well as presentations at the International Plant and Animal Genome conference, Poultry Science meetings, and the Poultry Breeders Roundtable. All SNP dat is available for public use through NCBI and the commercial release of a 60K SNP array (via Affymetrix). What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The goal of this project was to investigate turkey genome variation and to provide a resource for subsequent genomic work in the turkey by a wide sampling of populations for the development of a high-density single nucleotide polymorphism (SNP) chip with minimal ascertainment bias. Males from seven commercial lines, three heritage varieties and historical samples of wild turkeys from South Mexico, for a total of 11 turkey populations, were used for whole genome sequencing. After aligning against the turkey reference genome, 5.49 million SNPs were identified, which subsequently were used for the analysis of genetic diversity among the different populations. All commercial populations appear to share a common origin. The inclusion ofa wild turkey population allowed for the identification of the ancestral alleles for most of the SNPs. Six regions on five different turkey chromosomes (3, 4, 9, 14, and 22) showed differences between the wild and the domesticated populations with respect to ancestral and derived allelic states; derived alleles arise from a random DNA mutation event to produce a new allele that is different from the "original" or ancestral allele and, with selective breeding for desired traits, have been preferentially selected in domesticated animal populations. Domesticated populations showed the derived allelic state, while the wild populations showed the ancestral allelic state within these regions. The low level of genetic variation within those six regions, as well as the distinctness of the alleles in domesticated from wild populations, indicates the selection of specific allele combinations closely linked together on the same chromosome, that tend to be inherited together in domesticated populations. These data demonstrated the feasibility and potential utility of a turkey SNP chip. Subsequently, both primary turkey breeders provided DNA from additional birds and lines for further sequencing and SNP validation.In collaboration with Affymetrix, a 60K SNP array is now available for the first time for the turkey research community and commercial industry.

Publications

• Type: Journal Articles Status: Published Year Published: 2012 Citation: Aslam ML, Bastiaansen JW, Elferink MG, Megens HJ, Crooijmans RP, Blomberg le A, Fleischer RC, Van Tassell CP, Sonstegard TS, Schroeder SG, Groenen MA, Long JA. Whole genome SNP discovery and analysis of genetic diversity in Turkey (Meleagris gallopavo). BMC Genomics. 2012 Aug 14;13:391. doi: 10.1186/1471-2164-13-391.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Aslam ML, Bastiaansen JW, Megens HJ, Crooijmans RP, Nasreen F, Blomberg le A, Van Tassell CP, Sonstegard TS, Schroeder SG, Groenen MA, Long JA. Genome-wide candidate regions for selective sweeps revealed through massive parallel sequencing of DNA across ten turkey populations. BMC Genet. 2014 Nov 25;15:117. doi: 10.1186/s12863-014-0117-4.