1. 2008 Advances in DNA genotyping and sequencing genotyping and sequencing Tad S. Sonstegard USDA, ARS, Bovine Functional Genomics Laboratory BARC-East Beltsville, MD U.S.A. tad.sonstegard@ars.usda.gov SBTE XXII Aug. 22, 2008 Guaraja, Brazil

2. 2008 Past Genotyping and Sequencing - Livestock 8090001070 RFLP microsatellite SNP Sanger Next-Gen Gene SequenceESTMicroarraysDGE

3. 2008 Advances in genotyping

4. 2008 Where to advance from the 50K chip? Get Smaller –Low density multiplexing platform –Cost effective use of concentrated genotypic information Get Bigger –Higher density (300-600 K) Infinium II or Affymetrix type platform –More expensive platform for finer resolution mapping Linkage disequilibrium monogenic traits –Capture larger proportion of genetic diversity across cattle species in one platform

5. 2008 Goals of BovineSNP50 development develop an assay to surpass the minimum number of markers that would span the bovine genome at an average marker density of 100 kb achieve an average marker minor allele frequency (MAF) of at least 0.15 among common cattle breeds

6. 2008 MAF  taurus>composite>African>indicus Parentage SNP had significantly higher average MAF values Average MAF by Source/Breed Type

7. 2008 7 Phylogeny of Bovini

8. 2008 Conclusions from High-Res Hap Study Historic geographic ancestry better explains genotypic variation (and haplotype block structure) in cattle than does their more recent selection into breeds Estimated that it would be necessary to successfully assay >28,700 SNPs to construct an LD map for association studies >574,000 SNPs are needed to characterize the haplotype block structure across the entire bovine genome

9. 2008 Resources in Native Breeds PhenotypesMassiveLimitedVery Limited Genome Draft7X – SangerNoneNone 50K SNP chip51K Informative SNP>40K SNP>2.5 K SNP.4K AssayIn developmentNoneNone 300K chipNot enough SNP

10. 2008 SNP Discovery Pool ~15-35 gDNA samples for each library Cut with restriction enzyme and select 70-130 bp Sequence fragment ends 10-30X and cluster reads Identify clusters with SNPs (at least 2 alleles) Map to genomic coordinates and estimate MAF Design detection assays from genome sequence 3 libraries Holstein Angus Other beef 10 3 libraries Nelore Brahman Native Dairy

11. 2008 Where are we going? Testing of alternative breeds for WGS –Test use of 50K chip -> ~30,000 informative markers in Nelore –Genotyping for congenic line selection Evaluate and maintain genetic diversity –Pursue SNP discovery for indicine cattle using “next-gen” –Building a bigger chip? Chasing of IP/Biological knowledge –Genome partitioning technology for use on “next gen” Sequence 5-10 Mbp regions of genome –Sequencing Genomes of other breeds/ruminants –Defining genome activity –Single molecule sequencing “next next-gen” Defining true haplotypes Characterizing copy number variation Finding rare mutations Replacing genotyping - $100 Genome

12. 2008 The gap between genetic improvement and biology…

13. 2008 Systems & temporal framework: digestive tract 255 d Juvenile 180 d Fetus liver abomasumduodenum jejunum colon ileum cecum reticulum rumen spleen liver omasum abomasumduodenum jejunum ileum reticulum rumen omasum

14. 2008 Library Prep Process AAAAA cDNA synthesis TTTTT ___ Biotin Ligation of Adaptor NN Adaptor GATC CTAG Ligation of Adaptor AAAAA TTTTT ___Biotin Adaptor GATC CTAG MmeI Mme I Digestion Adaptor GATC CTAG MmeI NN Restriction Enzyme Digestion AAAAA TTTTT __Biotin GATC AAAAA mRNA Ready for clustering on Illumina GA flow cells

15. 2008

19. Differentially Expressed Gene http://animalgenomics.missouri.ed u 19

20. 2008 Differentially Expressed UTR http://animalgenomics.missouri.ed u 20

21. 2008 Future of functional genomics Wold - Nat. Genetics 2008

22. 2008

23. 23 USDA/CSREES/NRI2006-35616-166972006-35205-168882006-35205-16701USDA/ARS1265-31000-081D1265-31000-090-00D5438-31000-073D AIPL Laboratories Paul Van Raden George Wiggans Merial, Ltd Stewart Bauck Bull Studs/NAAB/Breed Associations ABS Global Accelerated Genetics Alta Genetics CRI/Genex Select Sires Semex Alliance Taurus Service HAUSAAJCA Funding Agencies/Collaborators BARC Curt Van Tassell Steven Schroeder Lakshmi Matukumalli Jeff O’Connell Larry Shade Alicia Bertles Missouri Jerry Taylor Robert Schnabel Jared Decker MARC Tim Smith Marc Allen Support Personnel Acknowledgements