Applied Bovine Genomics – “Delivering on the Promise” Ronnie D. Green National Program Leader Food Animal Production USDA / ARS

From gene to genome Gene – The functional and physical unit of heredity passed from parent to offspring Genome – The DNA comprising the complete genetic complement of an organism Genomics – a new scientific discipline of mapping, sequencing, and analyzing genomes

 3 billion base pairs (A, G, C, and T)  Every cell has two copies (alleles) of each chromosome  Estimated 30,000 to 40,000 genes (two copies or alleles of each gene)  Maybe 400,000 proteins produced A Mammalian Genome

First goal 300 markers Today > 21,000 markers Snelling et al. (2006)

Blackie Bonzo GENOME SCAN RESULTS IN ALL RESOURCE FAMILIES BTA2 MSTN BTA1 RPYD FATYD MARB, RPYD MARB BTA3 MARB, RPYD Fat yield BTA4 RPYD, FATYDWBS14 HCW, WBS3 BTA5 FAT, YG, RPYD, WBS14 RIBBONE, DP, RIBFAT, BWT BTA8 FAT, MARB FAT BTA15 WBS14 BTA27 MARB Dairy Form BTA29 WBS3, WBS14, Calpain BM PA BTA6 BWT, W365, HCW, LMA HCW, ADG

 Use additional DNA markers and animals to refine the location of the QTL to the QTN (gene mutation)  Use human and mouse mapping information (Comparative Mapping) Fine Mapping

 Leptin - fat deposition / DMI  DGAT – milk production  BHGR – milk components  Thyroglobulin - marbling  Calpastatin - tenderness  Calpain - tenderness  Somatostatin -- marbling Important genes affecting production traits

So…What Was Wrong with Our Approach?

Interagency Coordination Essential…. Interagency Working Group on Domestic Animal Genomics – Executive Office of the President Office of Science & Technology Policy (OSTP) National Science & Technology Council (NSTC) Committee on Science (co-Chairs NIH, NSF, OSTP) Established in winter 2002 Charter members – DOE, FDA, NIH, NSF, OMB, OSTP, USDA Chairperson -- Joseph Jen, USDA/REE Executive Director – Ronnie Green, USDA/ARS

Genomic Sequencing Launch: Dec Expected Finish: Spring 2007

International Collaboration – NIH / NHGRI -- $25M – State of Texas – $10M – USDA -- $11M – Australia -- $1M – Genome Canada -- $5M – New Zealand -- $1M – Beef Councils (US, TX, SD) – $0.82M Project Total = $53M

Bovine Genome Sequence – Final Assembly – v. 3.1 ~7.2-X coverage of the genome Total of 27.9M sequence reads Avg. trimmed read length - ~700 bp Size of the genome 2.87B base pairs N50 supercontig size - ~1M bp Used the International Integrated Map to order the assembly (Snelling et al., 2006) Released to GenBank and available through BCM web-site at:

Genome Sequence AGCTTTAAGCCATACCTTAG... GACATTACCTAGGAGCTTTAAGCCATAC AACTGGTCTACAG... GTTCAACGTCCTTGAC GTCGTAACCTGA... TCAACTGGTACA TCAACTGGTACGT... ACTTCCAGGAGACCTGTATC GTGCAACCACTGTATGCGA... AGTTGTGCCACGT ATCGTTCAAGTATGCGTAAATCGTTGT... ACGTAATAGTACGT GTCGTACATGT... TGACGTAACTGA GCCACATGTAGCGT... TATGCGTATGTGTAAACGTGGGTACTA AAACTACGTTGTTTACCAG... GTGGGACACTAGTGATCG TTAGACGATATCG... TATGACACGTACGT AATGTACACACACACACAC... ACGTGCGTCGT

Shotgun Assembly GCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC......AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC......AGCTTTAAGCCATACCTTAGGACATTACCTAGG...AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC... Consensus

Genome Project Objectives PHASE II – Tools and Annotation –Discovery of new genetic markers Single-nucleotide polymorphism markers Single genome in coverage from SNP discovery project –10,000 full length cDNA sequences Expressed pieces of the genome Characterize genome structure 4,000 completed thus far –75Mb finished sequence NIH Encode project – Ensembl automated annotation

Genetic Markers - Types Microsatellite –Based on differences in the number of short DNA sequences that are repeated at a specific point in the genome that can be detected using PCR –Also called simple sequence repeat (SSR) markers –Usually not associated with genes Single Nucleotide Polymorphisms (SNP) –Based upon simple changes of DNA at a specific point in the genome...CAGTATCACACACACACACACACACAGTCCTGAT......CAGTATCACACACACACACACACACACACACAGTCCTGAT......CCGTATCTAGGTAAATGTACAATCTTG......CCGTATCTAGGTATATGTACAATCTTG...

Phase II - SNP Project Expect 3 million SNP in the genome Light sequencing on each breed completed to discover SNPs Over 2M SNP found in the base Hereford sequence Holstein Jersey AngusLimousinBrahman Norwegian Red

SNP Discovery in the Genome Age...AGCTTTAAGCCATACCTTAGGACATTACCTAGG GCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC......AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC......AGCTTTAAGCCATACCTTAGGATATTACCTAGGAGCTTTAAGCCATAC... SNP Martha Dominette Consensus

SNP Discovery: Assembly

Phase II - SNP Project Current Status of Project: –Genotyping underway on total of 497 animals (19 breeds) for total of 39K SNP –(Breeds include: Angus, Hereford, Limousin, Charolais, Red Angus, Piedmontiese, Romagnola, Brahman, Santa Gertrudis, Beefmaster, Nellore, Gir, N’Dama, Sheko, Holstein, Jersey, Brown Swiss, Norwegian Red, Guernsey ) Bovine HapMap Consortium

Cost effective whole genome SNP genotyping …100K SNP for $200? 0.2 cents per genotype

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SNP Project Impacts Enhanced QTL Mapping –Hi density map will radically enhance mapping efforts Characterization of the Structure of the Genome –Haplotype blocks will advance understanding of how DNA information can be used Tree of Life –Identify evolutionary relationships among breeds, lines, species, etc. Reduced set of informative markers –“Whole-GENOME SELECTION”??

… to genomic selection. Cost-effective whole genome SNP genotyping Long-range LD in livestock Advanced statistical genetics (e.g. Meuwissen & Goddard) Genomic selection + + Could have major impact in animal and human genetics !

“We don’t need to know what the genes are” Gordon Dickerson, USDA-ARS US MARC (1995) "I don't actually care if it's making a protein or not, the equations are still the same.” Laurence Hurst, University of Bath (2006)

Haplotype From “haploid genotype.” A set of closely linked alleles (genes or DNA polymorphisms) inherited as a unit. Different combinations of polymorphisms are known as haplotypes.

LD Example – BTA1 MAF > 0.2 Holstein Limousin DGAT1TG

Tag SNP

Genetic Improvement -- SNP Based Whole Genome Selection UnknowngenesTagSNP Phenotypicdata EPD for unknownpolygenes SNPGenotypes (E)PD for identifiedHaplotypes “GenomeEnhanced”Selectioncriterion Molec. genetics Haplotype BLUP

Block “GE” PBV HaplotypeABCD Animal Animal

BFGL and AIPL -- BARC

The BeadStation detects the dye-labeled fragments and provides data as to which SNP markers are present at each of 1536 marker loci in each of 96 DNA samples. Using fluorescent dyes, 1536 different SNPs are assayed on the ends of each of 50,000 strands of 96 fiber optic cables. The Illumina BeadStation500G The Illumina GoldenGate™ Single Nucleotide Polymorphism (SNP) Assay Illumina Bead Array Matrix (96 fiber optic cables with 50,000 fibers each)

Project Overview - Goals Generate 50,000+ SNP assay platform Establish higher resolution HapMap –Identify haplotype blocks and TagSNP Demonstrate genome-wide selection works –Estimate variance components, develop models, etc –PREDICT GENETIC MERIT –Using Cooperative Dairy DNA Repository (CDDR) Fine map QTL and discover gene causations -- QTN

Post-Genome Sequencing …. What is the Rate-Limiting Step?

PHENOMICS……. Adaptability / Functionality???

 Production and production components  Type  Feed efficiency – expensive to measure  Reproduction- dissect components  Genetic resistance to disease  Select for multiple traits  Management “by genotype” – precision mgt Genomics Research

 PrP Gene – Scrapie, CWD, BSE?  FMD, Avian Influenza, PRRS  BRD, BVD, Johne’s  Mastitis resistance  New vaccine development  New drug targets / immunomodulators Genomics Research – Host- Pathogen Interaction

Forage Livestock Production? Bioenergy? Rumen Metagenomics?

X Gene Map Location QTL Map Location X The X-axis is the chromosomal map position of each gene in the expression array Generalized Results from a “Recombination Meets Function” Integrated Experiment The Y-axis is the chromosomal map position of the QTL that explains the most variation in expression levels of each gene in the expression array “Genetical Genomics” D. Pomp, 2006 EU-US

X Gene Map Location QTL Map Location X Expressed genes regulated by linked (cis-acting) QTL May immediately reveal identity of QTL D. Pomp, 2006 EU-US

X Gene Map Location QTL Map Location X Expressed genes regulated by linked (cis-acting) QTL Groups of unlinked expressed genes that are regulated by a single QTL May immediately reveal master regulators and key pathways, networks D. Pomp, 2006 EU-US

X Gene Map Location QTL Map Location X Expressed genes regulated by linked (cis-acting) QTL Groups of unlinked expressed genes that are regulated by a single QTL Expressed genes regulated by unlinked (trans-acting) QTL May identify inclusive pathway or network for a QTL, and potential epistatic interactions D. Pomp, 2006 EU-US

Systems Biology: The Legacy of Quantitative Genetics? Instead of analyzing individual components or aspects of the organism, systems biologists focus on all the components and the interactions among them, all as part of one system. The interactions of numerous genes, proteins, mechanisms and the organism's external environment, produce an individual’s phenotype.

SAVAGE, Md. and MINNEAPOLIS - June 11, 2002 MetaMorphix signs deal to develop genetic selection tool using cattle genome -- Exclusive agreement with Cargill's Caprock Cattle Feeders and Excel Corporation expected to result in superior beef for consumers

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