Applied Bovine Genomics

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Applied Bovine Genomics Delivering on the
Promise

• Ronnie D. Green
• National Program Leader
• Food Animal Production
• USDA / ARS

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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
• 1986 -- a new scientific discipline of mapping,
  sequencing, and analyzing genomes

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A Mammalian Genome

• 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

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Snelling et al. (2006)
First goal 300 markers Today gt 21,000 markers
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HCW, ADG
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Fine Mapping

• 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)

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Important genes affecting production traits

• Leptin - fat deposition / DMI
• DGAT milk production
• BHGR milk components
• Thyroglobulin - marbling
• Calpastatin - tenderness
• Calpain - tenderness
• Somatostatin -- marbling

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SoWhat Was Wrong with Our Approach?
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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

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Genomic Sequencing
Launch Dec. 2003 Expected Finish Spring 2007
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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

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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
• www.hgsc.bcm.tmc.edu/projects/bovine

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Genome Sequence
AGCTTTAAGCCATACCTTAG . . . GACATTACCTAGGAGCTTTAAGC
CATAC
AATGTACACACACACACAC . . . ACGTGCGTCGT
AACTGGTCTACAG . . . GTTCAACGTCCTTGAC
ATCGTTCAAGTATGCGTAAATCGTTGT . . . ACGTAATAGTACGT
GTCGTAACCTGA . . . TCAACTGGTACA
GTCGTACATGT . . . TGACGTAACTGA
TCAACTGGTACGT . . . ACTTCCAGGAGACCTGTATC
GCCACATGTAGCGT . . . TATGCGTATGTGTAAACGTGGGTACTA
GTGCAACCACTGTATGCGA . . . AGTTGTGCCACGT
AAACTACGTTGTTTACCAG . . . GTGGGACACTAGTGATCG
TTAGACGATATCG . . . TATGACACGTACGT
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Shotgun Assembly
...AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATA
C...
...AGCTTTAAGCCATACCTTAGGACATTACCTAGG
...AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATA
C...
GCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC...
...AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATA
C...
...AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATA
C...
Consensus
...AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATA
C...
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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

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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

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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
Norwegian Red
Angus
Limousin
Brahman
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SNP Discovery in the Genome Age
Dominette
...AGCTTTAAGCCATACCTTAGGACATTACCTAGG
GCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC... ...AGC
TTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC... .
..AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC
... ...AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGC
CATAC... ...AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTT
TAAGCCATAC...
Consensus
...AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATA
C...
...AGCTTTAAGCCATACCTTAGGATATTACCTAGGAGCTTTAAGCCATA
C...
Martha
SNP
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SNP Discovery Assembly
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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, NDama,
  Sheko, Holstein, Jersey, Brown Swiss, Norwegian
  Red, Guernsey)

Bovine HapMap Consortium
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Cost effective whole genome SNP genotyping 100K
SNP for 200?
0.2 cents per genotype
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????????????????
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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??

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to genomic selection.

• Cost-effective whole genome
• SNP genotyping
• Long-range LD in livestock
• Advanced statistical genetics
• (e.g. Meuwissen Goddard)

Could have major impact in animal and human
genetics !
Genomic selection
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We dont 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)
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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.

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LD Example BTA1 MAF gt 0.2
Holstein
DGAT1
TG
Limousin
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Tag SNP
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Genetic Improvement -- SNP Based Whole Genome
Selection
EPD for unknown polygenes
Phenotypic data
Unknown genes TagSNP
Genome Enhanced Selection criterion
Molec. genetics
Haplotype BLUP
(E)PD for identified Haplotypes
SNP Genotypes
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Block Block Block Block GE PBV
Haplotype A B C D GE PBV
1 0.01 1.03 -1.23 6.35
2 0.06 -0.74 0.98 2.19
3 0.05
4 -8.59
Animal 1 1 1 1 2 2 2 1 3
Animal 1 0.01 0.01 1.03 -0.74 0.98 0.98 6.35 0.05 8.67
Animal 2 2 2 1 1 2 2 2 4
Animal 2 0.06 0.06 1.03 1.03 0.98 0.98 2.19 -8.59 -2.26
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BFGL and AIPL -- BARC
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The Illumina GoldenGate Single Nucleotide
Polymorphism (SNP) Assay
Illumina Bead Array Matrix (96 fiber optic
cables with 50,000 fibers each)
The Illumina BeadStation500G
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.
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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

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Post-Genome Sequencing .
What is the Rate-Limiting Step?
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PHENOMICS.
Adaptability / Functionality???
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Genomics Research

• 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

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Genomics Research Host-Pathogen Interaction

• PrP Gene Scrapie, CWD, BSE?
• FMD, Avian Influenza, PRRS
• BRD, BVD, Johnes
• Mastitis resistance
• New vaccine development
• New drug targets / immunomodulators

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Forage Livestock Production?
Bioenergy? Rumen Metagenomics?
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Genetical Genomics
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
X 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5
4 3 2 1
Generalized Results from a Recombination Meets Fu
nction Integrated Experiment
QTL Map Location
The X-axis is the chromosomal map position of
each gene in the expression array
Gene Map Location
D. Pomp, 2006 EU-US
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X 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5
4 3 2 1
May immediately reveal identity of QTL
QTL Map Location
Gene Map Location
Expressed genes regulated by linked (cis-acting)
QTL
D. Pomp, 2006 EU-US
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X 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5
4 3 2 1
May immediately reveal master regulators and key
pathways, networks
QTL Map Location
Gene Map Location
Expressed genes regulated by linked (cis-acting)
QTL Groups of unlinked expressed genes that are
regulated by a single QTL
D. Pomp, 2006 EU-US
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May identify inclusive pathway or network for a
QTL, and potential epistatic interactions
X 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5
4 3 2 1
QTL Map Location
Gene Map Location
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
D. Pomp, 2006 EU-US
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• 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
  individuals phenotype.

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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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