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– 1986 -- 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 markersToday > 21,000 markers

Snelling et al. (2006)

BlackieBonzo

GENOME SCAN RESULTS IN ALL RESOURCE FAMILIES

BTA2

MSTN

BTA1

RPYDFATYD MARB, RPYD

MARB

BTA3

MARB, RPYDMARB, RPYDFat yield

BTA4

RPYD, FATYDWBS14

HCW, WBS3

BTA5

FAT, YG, RPYD, WBS14RIBBONE, DP, RIBFAT, BWT

BTA8

FAT, MARBFAT

BTA15

WBS14

BTA27

MARBDairy 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. 2003Expected 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: www.hgsc.bcm.tmc.edu/projects/bovine

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...

...AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC...

...AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC...

...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

Angus Limousin Brahman Norwegian Red

SNP Discovery in the Genome Age

...AGCTTTAAGCCATACCTTAGGACATTACCTAGG GCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC......AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC......AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC......AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC......AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC...

...AGCTTTAAGCCATACCTTAGGACATTACCTAGGAGCTTTAAGCCATAC...

...AGCTTTAAGCCATACCTTAGGATATTACCTAGGAGCTTTAAGCCATAC...

SNPMartha

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

????????????????

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 areWe don’t need to know what the genes are”” Gordon Dickerson, USDA-ARS US MARC (1995)Gordon Dickerson, USDA-ARS US MARC (1995)

"I don't actually care if it's making a protein or not, "I don't actually care if it's making a protein or not, the equations are still the same.”the equations are still the same.”

Laurence Hurst, University of Bath (2006)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

DGAT1 TG

Tag SNP

Genetic Improvement -- SNP Based Whole Genome SelectionGenetic Improvement -- SNP

Based Whole Genome Selection

UnknownUnknowngenesgenes

TagSNPTagSNP

PhenotypicPhenotypicdatadata

EPD forEPD for

unknownunknownpolygenespolygenes

SNP SNP GenotypesGenotypes

(E)PD for(E)PD for

identifiedidentifiedHaplotypesHaplotypes

““GenomeGenomeEnhanced”Enhanced”SelectionSelectioncriterioncriterion

Molec. geneticsMolec. genetics Haplotype BLUPHaplotype BLUP

Block“GE”

PBVHaplotype A B C D

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

0.01

+0.01

1.03

-0.74

0.98

+0.98

6.35

+0.058.67

Animal 2

2 2 1 1 2 2 2 4

0.06

+0.06

1.03

+1.03

0.98

+0.98

2.19

-8.59-2.26

BFGL and AIPL -- BARC

The BeadStation detects the dye-labeledfragments 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?

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The X-axis is the chromosomal map position The X-axis is the chromosomal map position of each gene in the expression arrayof each gene in the expression array

GeneralizedResults from a

“RecombinationMeets

Function”Integrated

Experiment

The Y-axis is the The Y-axis is the chromosomal chromosomal

map position of map position of the QTL that the QTL that explains the explains the

most variation in most variation in expression levels expression levels of each gene in of each gene in the expression the expression

arrayarray

“Genetical Genomics”

D. Pomp, 2006 EU-US

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Expressed genes regulated by linked (cis-acting) QTLExpressed genes regulated by linked (cis-acting) QTL

May May immediately immediately

revealrevealidentity of identity of

QTL QTL

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Expressed genes regulated by linked (cis-acting) QTLExpressed genes regulated by linked (cis-acting) QTLGroups of unlinked expressed genes that are regulated by a single QTLGroups of unlinked expressed genes that are regulated by a single QTL

May May immediately immediately

revealrevealmaster master

regulators regulators and key and key

pathways, pathways, networksnetworks

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Expressed genes regulated by linked (cis-acting) QTLExpressed genes regulated by linked (cis-acting) QTLGroups of unlinked expressed genes that are regulated by a single QTLGroups of unlinked expressed genes that are regulated by a single QTLExpressed genes regulated by unlinked (trans-acting) QTLExpressed genes regulated by unlinked (trans-acting) QTL

May May identify identify inclusive inclusive

pathway or pathway or networknetwork

for a QTL, for a QTL, and and

potential potential epistatic epistatic

interactionsinteractions

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

h