1. J. B. Cole Animal Improvement Programs Laboratory Agricultural Research Service, USDA Beltsville, MD 20705-2350, USA john.cole@ars.usda.gov 2013 Genomic selection and systems biology – lessons from dairy cattle breeding

2. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (2) Cole Dairy Cattle l 9 million cows in US l Attempt to have a calf born every year l Replaced after 2 or 3 years of milking l Bred via AI l Bull semen collected several times/week. Diluted and frozen l Popular bulls have 10,000+ progeny l Cows can have many progeny though super ovulation and embryo transfer

3. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (3) Cole Data Collection l Monthly recording w Milk yields w Fat and Protein percentages w Somatic Cell Count (Mastitis indicator) l Visual appraisal for type traits l Breed Associations record pedigree l Calving difficulty and Stillbirth

4. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (4) Cole Traditional evaluations 3X/year l Yield w Milk, Fat, Protein l Type w Stature, Udder characteristics, feet and legs l Calving w Calving Ease, Stillbirth l Functional w Somatic Cell, Productive Life, Fertility

5. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (5) Cole Use of evaluations l Bulls to sell semen from l Parents of next generation of bulls l Cows for embryo donation

6. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (6) Cole Parents Selected Dam Inseminated Embryo Transferred to Recipient Bull Born Semen collected (1yr) Daughters Born (9 m later) Daughters have calves (2yr later) Bull Receives Progeny Test (5 yrs) Lifecycle of bull Genomic Test

7. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (7) Cole Benefit of genomics l Determine value of bull at birth l Increase accuracy of selection l Reduce generation interval l Increase selection intensity l Increase rate of genetic gain

8. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (8) Cole History of genomic evaluations l Dec. 2007BovineSNP50 BeadChip available l Apr. 2008First unofficial evaluation released l Jan. 2009Genomic evaluations official for Holstein and Jersey l Aug. 2009Official for Brown Swiss l Sept. 2010Unofficial evaluations from 3K chip released l Dec. 20103K genomic evaluations to be official l Sept. 2011 Infinium BovineLD BeadChip available

9. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (9) Cole Cattle SNP Collaboration - iBMAC l Develop 60,000 Bead Illumina iSelect® assay w USDA-ARS Beltsville Agricultural Research Center: Bovine Functional Genomics Laboratory and Animal Improvement Programs Laboratory w University of Missouri w University of Alberta w USDA-ARS US Meat Animal Research Center l Started w/ 60,800 beads – 54,000 useable SNP

10. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (10) Cole Participants  Illumina  Marylinn Munson  Cindy Lawley  Christian Haudenschild  BARC  Curt Van Tassell  Lakshmi Matukumalli  Tad Sonstegard  Missouri  Jerry Taylor  Bob Schnabel  Stephanie McKay  Alberta  Steve Moore  USMARC – Clay Center  Tim Smith  Mark Allan  USDA/NRI/CSREES  2006-35616-16697  2006-35205-16888  2006-35205-16701  USDA/ARS  1265-31000-081D  1265-31000-090D  5438-31000-073D  Merial  Stewart Bauck  NAAB  Godon Doak  ABS Global  Accelerated Genetics  Alta Genetics  CRI/Genex  Select Sires  Semex Alliance  Taurus Service iBMAC Consortium Funding Agencies

11. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (11) Cole Use of HD l Currently only 50K subset of SNP used l Some increase in accuracy from better tracking of QTL possible l Potential for across breed evaluations l Requires few new HD genotypes once adequate base for imputation developed

12. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (12) Cole LD chip l 6909 SNP mostly from SNP50 chip w 9 Y Chr SNP included for sex validation w 13 Mitochondrial DNA SNP w Evenly spaced across 30 Chr (increased density at ends) l Developed to address performance issues with 3K while continuing to provide low cost genotyping l Provides over 98% accuracy imputing 50K genotypes l Included beginning with Nov genomic evaluation

13. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (13) Cole Development of LD chip l Consortium included researchers from USA, AUS and FRA l Objective: good imputation performance in dairy breeds w Uniform distribution except heavier at chromosome ends w High MAF, avg MAF over 30% for most breeds w Adequate overlap with 3K

14. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (14) Cole Genomic evaluation program steps l Identify animals to genotype l Sample to lab l Genotype sample l Genotype to USDA l Calculate genomic evaluation l Release monthly

15. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (15) Cole Responsibilities of requester l Insure animal is properly identified eg HOCANF000123456789 l Enroll animal with breed association or insure pedigree on animal and dam reaches AIPL l Collect clean, clearly labeled DNA sample l Get sample to lab in time to be included in desired month’s results l Resolve parentage conflicts quickly

16. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (16) Cole Steps to prepare genotypes l Nominate animal for genotyping l Collect blood, hair, semen, nasal swab, or ear punch w Blood may not be suitable for twins l Extract DNA at laboratory l Prepare DNA and apply to BeadChip l Do amplification and hybridization, 3-day process l Read red/green intensities from chip and call genotypes from clusters

17. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (17) Cole What can go wrong l Sample does not provide adequate DNA quality or quantity l Genotype has many SNP that can not be determined (90% call rate required) l Parent-progeny conflicts w Pedigree error w Sample ID error (Switched samples) w Laboratory error w Parent-progeny relationship detected that is not in pedigree

18. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (18) Cole Lab QC l Each SNP evaluated for w Call Rate w Portion Heterozygous w Parent-progeny conflicts l Clustering investigated if SNP exceeds limits l Number of failing SNP is indicator of genotype quality l Target fewer than 10 SNP in each category

19. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (19) Cole Before clustering adjustment 86% call rate

20. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (20) Cole After clustering adjustment 100% call rate

21. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (21) Cole Parentage validation and discovery l Parent-progeny conflicts detected w Animal checked against all other genotypes w Reported to breeds and requesters w Correct sire usually detected l Maternal Grandsire checking w SNP at a time checking w Haplotype checking more accurate l Breeds moving to accept SNP in place of microsatellites

22. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (22) Cole Checking facility l Labs place genotype files on AIPL server l Genotypes run through analysis procedures, but not added to database l Reports on missing nominations and QC data returned to Lab l Lab can w Detect sample misidentification w Improve clustering w Apply the same checks used by AIPL

23. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (23) Cole Imputation l Based on splitting the genotype into individual chromosomes (maternal & paternal contributions) l Missing SNP assigned by tracking inheritance from ancestors and descendents l Imputed dams increase predictor population l 3K, LD, & 50K genotypes merged by imputing SNP not on LD or 3K

24. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (24) Cole Recessive defect discovery l Check for homozygous haplotypes l Most haplotype blocks ~5Mbp long l 7 – 90 expected, but 0 observed l 5 of top 11 haplotypes confirmed as lethal l Investigation of 936 – 52,449 carrier sire  carrier MGS fertility records found 3.0 – 3.7% lower conception rates

25. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (25) Cole Breed BTA chromo- some Location, Mbases Carrier frequency, % Holstein 562–68 4.5 193–98 4.6 892–97 4.7 Jersey1511–1623.4 Brown Swiss 742–4714.0 Haplotypes impacting fertility

26. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (26) Cole Collaboration l Full sharing of genotypes with Canada w CDN calculates genomic evaluations on Canadian base l Trading of Brown Swiss genotypes with Switzerland, Germany, and Austria w Interbull may facilitate sharing l Agreements with Italy and Great Britain provide genotypes for Holstein w Negotiations underway with other countries

27. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (27) Cole Calculation of genomic evaluations l Deregressed values derived from traditional evaluations of predictor animals l Allele substitutions random effects estimated for 45,187 SNP l Polygenic effect estimated for genetic variation not captured by SNP l Selection Index combination of genomic and traditional not included in genomic l Applied to yield, fitness, calving and type traits

28. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (28) Cole Reliabilities for young Holsteins* *Animals with no traditional PTA in April 2011 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 404550556065707580 Reliability for PTA protein (%) Number of animals 3K genotypes 50K genotypes

29. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (29) Cole Use of genomic evaluations l Determine which young bulls to bring into AI service l Use to select mating sires l Pick bull dams l Market semen from 2-year-old bulls

30. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (30) Cole Use of LD genomic evaluations l Sort heifers for breeding w Flush w Sexed semen w Beef bull l Confirm parentage to avoid inbreeding l Predict inbreeding depression better l Precision mating considering genomics (future)

31. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (31) Cole Ways to increase accuracy l Automatic addition of traditional evaluations of genotyped bulls when reach 5 years of age l Possible genotyping of 10,000 bulls with semen in repository l Collaboration with more countries l Use of more SNP from HD chips l Full sequencing – Identify causative mutations

32. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (32) Cole Application to more traits l Animal’s genotype is good for all traits l Traditional evaluations required for accurate estimates of SNP effects l Traditional evaluations not currently available for heat tolerance or feed efficiency l Research populations could provide data for traits that are expensive to measure l Will resulting evaluations work in target population?

33. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (33) Cole Impact on producers l Young-bull evaluations with accuracy of early 1st­crop evaluations l AI organizations marketing genomically evaluated 2- year-olds l Genotype usually required for cow to be bull dam l Rate of genetic improvement likely to increase by up to 50% l Studs reducing progeny-test programs

34. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (34) Cole Summary l Extraordinarily rapid implementation of genomic evaluations l Chips provide genotypes of high accuracy l Comprehensive checking insures quality of genotypes stored l Young-bull acquisition and marketing now based on genomic evaluations l Genotyping of many females because of lower cost low density chips

35. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (35) Cole Why genomics works in dairy l Extensive historical data available l Well-developed genetic evaluation program l Widespread use of AI sires l Progeny test programs l High valued animals, worth the cost of genotyping l Long generation interval which can be reduced substantially by genomics

36. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (36) Cole History of genomic evaluations l Dec. 2007BovineSNP50 BeadChip available l Apr. 2008First unofficial evaluation released l Jan. 2009Genomic evaluations official for Holstein and Jersey l Aug. 2009Official for Brown Swiss l Sept. 2010Unofficial evaluations from 3K chip released l Dec. 20103K genomic evaluations to be official l Sept. 2011 Infinium BovineLD BeadChip available

37. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (37) Cole Current sources of data AIPLCDCB NAAB PDCA DHI Universities AIPL Animal Improvement Programs Lab., USDA CDCBCouncil on Dairy Cattle Breeding DHIDairy Herd Improvement (milk recording organizations) NAABNational Association of Animal Breeders (AI) PDCAPurebred Dairy Cattle Association (breed registries)

38. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (38) Cole Sources of genomic data Genomic Evaluation Lab Requester (Ex: AI, breeds) Dairy producers DNA laboratories samples genotypes nominations evaluations

39. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (39) Cole How does genetic selection work? l ΔG = genetic gain each year reliability = how certain we are about our estimate of an animal’s genetic merit (genomics can  ) selection intensity = how “picky” we are when making mating decisions (management can  ) l genetic variance = variation in the population due to genetics (we can’t really change this) generation interval = time between generations (genomics can  )

40. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (40) Cole Calculation of genomic evaluations l Deregressed values derived from traditional evaluations of predictor animals l Allele substitutions random effects estimated for 45,187 SNP l Polygenic effect estimated for genetic variation not captured by SNP l Selection Index combination of genomic and traditional not included in genomic l Applied to yield, fitness, calving, and type traits

41. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (41) Cole Genetic merit of Jersey bulls Breeding Year Net Merit ($)

42. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (42) Cole What is a SNP genotype worth? For the protein yield (h 2 =0.30), the SNP genotype provides information equivalent to an additional 34 daughters Pedigree is equivalent to information on about 7 daughters

43. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (43) Cole And for daughter pregnancy rate (h 2 =0.04), SNP = 131 daughters What is a SNP genotype worth?

44. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (44) Cole Holstein prediction accuracy Trait a Bias b bREL (%)REL gain (%) Milk (kg)−64.30.9267.128.6 Fat (kg)−2.70.9169.831.3 Protein (kg) 0.70.8561.523.0 Fat (%) 0.01.0086.548.0 Protein (%) 0.00.9079.040.4 PL (months)−1.80.9853.021.8 SCS 0.00.8861.227.0 DPR (%) 0.00.9251.221.7 Sire CE 0.80.7331.010.4 Daughter CE−1.10.8138.419.9 Sire SB 1.50.9221.8 3.7 Daughter SB− 0.20.8330.313.2 a PL=productive life, CE = calving ease and SB = stillbirth. b 2011 deregressed value – 2007 genomic evaluation.

45. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (45) Cole Many chips are available l BovineSNP50 w Version 1 54,001 SNP w Version 2 54,609 SNP w 45,187 used in evaluations l HD w 777,962 SNP w Only 50K SNP used, w >1700 in database l LD w 6,909 SNP w Replaced 3K HD 50KV2 LD

46. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (46) Cole Genotypes and haplotypes l Genotypes indicate how many copies of each allele were inherited l Haplotypes indicate which alleles are on which chromosome l Observed genotypes partitioned into the two unknown haplotypes w Pedigree haplotyping uses relatives w Population haplotyping finds matching allele patterns

47. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (47) Cole O-Style Haplotypes Chromosome 15

48. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (48) Cole Haplotyping program – findhap.f90 l Begin with population haplotyping w Divide chromosomes into segments, ~250 to 75 SNP / segment w List haplotypes by genotype match w Similar to fastPhase, IMPUTE l End with pedigree haplotyping w Detect crossover, fix noninheritance w Impute nongenotyped ancestors

49. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (49) Cole Recessive defect discovery l Check for homozygous haplotypes w 7 to 90 expected but none observed w 5 of top 11 are potentially lethal w 936 to 52,449 carrier sire-by-carrier MGS fertility records w 3.1% to 3.7% lower conception rates w Some slightly higher stillbirth rates l Confirmed Brachyspina same way

50. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (50) Cole We’re working on new tools Cole, J.B., and Null, D.J. 2012. AIPL Research Report GENOMIC2: Use of chromosomal predicted transmitting abilities. Available: http://aipl.arsusda.gov/reference/chromosomal_pta_query.html. http://aipl.arsusda.gov/reference/chromosomal_pta_query.html

51. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (51) Cole Impact on producers l Young-bull evaluations with accuracy of early 1st­crop evaluations l AI organizations marketing genomically evaluated 2- year-olds l Genotype usually required for cow to be bull dam l Rate of genetic improvement likely to increase by up to 50% l Studs reducing progeny-test programs

52. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (52) Cole l Expected value of Mendelian sampling no longer equal to 0 l Key assumption of animal models l References: w Patry, Ducrocq 2011 GSE 43:30 w Vitezica et al 2011 Genet Res (Camb) pp. 1–10. Bias from Pre-Selection

53. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (53) Cole l Bulls born in 2008, progeny tested in 2009, with daughter records in 2012, were pre-selected: w 3,434 genotyped vs. 1,096 sampled w Now >10 genotyped per 1 marketed l Potential for bias: w 178 genotyped progeny w 32 sons progeny tested Pre-Selection Bias Now Beginning

54. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (54) Cole l 1-Step to incorporate genotypes w Flexible models, many recent studies w Foreign data not yet included l Multi-step GEBV, then insert in AM w Same trait (Ducrocq and Liu, 2009) w Or correlated trait (Mantysaari and Stranden, 2010; Stoop et al, 2011) w Foreign genotyped bulls included Methods to Reduce Bias

55. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (55) Cole Gene set enrichment analysis-SNP Gene pathways (G) GWAS results Score increase is proportional to SNP test statistic Nominal p-value corrected for multiple testing Pathways with moderate effects Holden et al., 2008 (Bioinformatics 89:1669-1683. doi:10.2527/jas.2010-3681) SNP ranked by significance (L) SNP in pathway genes (S) Score increases for each L i in S Permutation test and FDR Includes all SNP, S, that are included in L The more SNP in S that appear near the top of L, the higher the Enrichment Score

56. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (56) Cole Adaptive weight matrix l Need to add this

57. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (57) Cole We hope to identify regulatory networks Fortes et al., 2011 (J. Animal Sci. 89:1669-1683. doi:10.2527/jas.2010-3681) Candidate genes and pathways that affect age at puberty common to both breeds

58. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (58) Cole Network analysis Fortes et al., 2011 (J. Animal Sci. 89:1669-1683. doi:10.2527/jas.2010-3681) Gene network – the red center identifies highly connected nodes. Subnetwork of interacting transcription factors from the puberty network. Subnetwork of interacting transcription factors from a collection of mouse and human data. (Validation step.)

59. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (59) Cole Enriched pathways Fortes et al., 2011 (J. Animal Sci. 89:1669-1683. doi:10.2527/jas.2010-3681)

60. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (60) Cole Transcription factor network Fortes et al., 2011 (J. Animal Sci. 89:1669-1683. doi:10.2527/jas.2010-3681) Yellow genes were submitted to database. Other nodes were mined from FunCoup. Red: protein- protein interaction Blue: mRNA coexpression

61. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (61) Cole GWAS for birth weight PTA h Cole et al.(2013), unpublished data

62. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (62) Cole KEGG pathways for birth weight l Waiting on DMB

63. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (63) Cole We have divergent populations Cole et al., 2005 (J. Dairy Sci. 88(4):1529–1539)

64. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (64) Cole What can we learn from this? l We are not going to find big QTL l We may identify gene networks affecting complex phenotypes l We’re going to learn how much we don’t know about functional genomics in the cow

65. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (65) Cole Conclusions l…l…

66. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (66) Cole Acknowledgments l…l…

67. Keygene N.V., Wageningen, The Netherlands, 29 May 2013 (67) Cole Questions? http://gigaom.com/2012/05/31/t-mobile-pits-its-math-against-verizons-the-loser-common-sense/shutterstock_76826245/