6/14/13. Jerry Taylor Emerging Technologies Committee Breakout. Bovine Respiratory Disease. Respiratory Disease. The Rumors Are True

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1 The Rumors Are True Updates on USDA Bovine Respiratory Disease, Feed Efficiency and Heifer Fertility Projects Jerry Taylor University of Missouri Beef Improvement Federation Annual Meeting Renaissance Hotel and Convention Center, Oklahoma City, June, 203 Respiratory Disease 2 Bovine Respiratory Disease S. McGuirk s Diagnostic Criteria 3 Identify genomic regions associated with BRD resistance/susceptibility in beef and dairy cattle Population A Dairy Calves Population A 2000 Holstein calves Samples on 203 calves collected and clinical scores obtained Population B 00 Holstein replacement heifers Diagnostics for Mycoplasma, P. Multocida, M. Haemolytica, H. Somni, Bovine respiratory syncytial virus, Bovine viral diarrhea virus, IBR completed Population C 2000 Bos taurus feedlot cattle Population D 00 Bos taurus purebred bulls DA extracted, and genotyped for 77,000 SPs 5 6

2 GWAS Competing Models Common Associations GBLUP PLIK Purple = GBLUP Red = PLIK Green = SVS7 SVS7 (Golden Helix) 7 M Replacement Holstein Heifers GBLUP Heritabilities Samples on 00 heifers collected and clinical scores obtained BRD (2%) asal discharge (9%) Total clinical score (%) Diagnostics for Mycoplasma, P. Multocida, M. Haemolytica, H. Somni, bovine respiratory syncytial virus, bovine viral diarrhea virus, IBR completed Pasturella multocida (2%) Mycoplasma (2%) Heritabilities similar for BRD Case-Control BRSV (6%) BUT ρg between SP effects in CA and M = 0 Mannheimia haemolytica (%) BRD is a mixture of diseases and pathogens differ between M and CA Cough (%) Temperature (%) DA extracted, and 77,000 SPs genotyped Ocular discharge/ear tilt (%) 9 Identify Sires of these Calves Challenge Study ~70% of Holstein calves sire identified All AI Holstein bulls are genotyped with the SP50 or HD We can use these genotypes to: a) Match bulls to calves b) Predict the MBVs of sires of these calves to BRD susceptibility Mean MBV for BRD Risk = kg Angus calves challenged with Mycoplasma bovis, P. Multocida, M. Haemolytica, Bovine viral diarrhea virus, and Bovine respiratory syncytial virus (=2 in 20 and = in 202) Mean MBV for BRD Risk = +2. Identify genes expressed with given pathogens via RAseq of bronchia-alveolar lymph nodes Characterize immunological responses BRD Unaffected Calves BRD Affected Calves 2 2

3 Bronchial Lymph odes Analysis Using Tuxedo Suite 3 Metagenomics/Boogergenomics: What Pathogens Cause BRD? 20 =2 Data 2 CTL vs All 2 CTL vs BACTERIA 2 CTL vs 6 VIRUS 5 Metagenomics: What Pathogens Cause BRD? Feed Efficiency 7 3

4 Genomic Selection Four Data Sets (=5,02) Breed SP50 HD #Animals #SPs 203 Animals 2 Total Animals 2 Angus,093 5,603 77, ,03 Charolais 2 2 /A 2 Charolais Ang Commercial Xbred Gelbvieh /A 369 Hereford ,5 300,52 Limousin , ormande 3 3 Pied Ang Simm /A 236 Red Angus ,7 3 5 Simm Ang 2, ,0 690, 909 3,79 Wagyu /A 50 5 Total 3,9 2,2 6,5 2,55,666 Imputed using Beagle 2 Does not include the possibility of a data swap with TEASUG, the Canadian Feed Efficiency ConsorEum or USMARC animals Hereford Cattle fed at Olsens (HD) 7 animals in contemporary groups F 2 composites fed at USMARC (50K),0 animals in 5 contemporary groups Legacy Simmental cattle fed at Illinois (HD), animals in 202 contemporary groups Legacy Angus fed at Circle A (HD) and Angus fed at MU,50 animals in 2 contemporary groups 20 Dry Matter Intake Mid-Test Metabolic Weight 2 22 Average Daily Gain RFI 23 2

5 Large Effect QTLs Large Effect QTLs /Users/dorian/Desktop/FeedEffic/summary Saved: 3/3/3 9:3:25 AM Genomic Selection 36 Page of Printed For: Dorian Garrick Dry Matter Intake Mid-test Metab Wt Brd #SP %Var chr_mb SP %Var chr_mb AA _ _23 AA _ _0 AA _ _3 AA 2.6 _ _9 AA _ _ AA _ _3 AA _ _70 AA _ _6 HER _ _ HER _ _93 HER _ _63 HER _ _70 HER _ _ HER _ _25 HER _ _3 HER _ _2 SIM _ _2 SIM _ _6 SIM _ _73 SIM _ _7 SIM _ 2.0 _50 SIM _ _59 SIM _ _25 SIM _ _ _ _ _ _ _. _ _ _ _ _ _ _ _ _ _ _ _ _2 Gain on Test ResidFeed Intake SP %Var chr_mb SP %Var chr_mb _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 2.95 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 26 Methane and Feed Efficiency αˆ =(2 piqi ) Μ'G u i RelaEonship between RFI and methane produceon in steers (lei) and heifers (right) 27 Heifer Fertility 2 Sequencing Cost Fearless Leader Budget Management Sparky Extension Web-based simulation Economic Modeling Budget Mismanagement Selection Indices I warned J-Rod Brian Kinghorn Communications (aka the really really smart guy) Megan is OK

6 Whole Genome Sequencing Deletion Mapping You are a nobody in genomics until you sequence your own dog... Miranda has a 37 bp deletion which includes the last exon (exon ) of FA 3 This is a Loss of Function Allele 32 Let s Sequence Some (Angus) Bulls Bar Emulation EXT FS FS C FS FS C FS FS C GDAR SVF Traveler 23D ame Registration Coverage Inbreeding Description supported by only single read probably seq e rror coverage >XX l ikely repetitive e lement or misaligned read insertions supported by only a single read deletions supported by only a single read genotype '- ' these are the i ndividually deleted bases for multibase deletions which are group - 23 low coverage SPs supported by only single read but HQ position i n at l east other sample low coverage SPs supported by only single read AD skewed freq but HQ position i n at l east other sample complicated SPs with i ndels based on a single read complicated i ndels i nvolving multiple bases HOMOzygous splice site +/- bp CDS HETEROzygous splice site +/- bp CDS HOMOzygous splice site +/- 2bp CDS HETEROzygous splice site +/- 2bp CDS HOMOzygous within UTR HETEROzygous within UTR HOMOzygous deletions HOMOzygous i nsertions HOMOzygous i ndels that i nsert/delete AA HOMOzygous i ndels that cause frameshifts i n genes HOMOzygous i ndels within genic regions HETEROzygous deletions HETEROzygous deletions that delete AA HETEROzygous deletions that cause frameshifts i n genes HETEROzygous deletions within genic regions skewed HETEROzygous deletions skewed HETEROzygous deletions that delete AA skewed HETEROzygous deletions that cause frameshifts i n genes skewed HETEROzygous deletions within genic regions HETEROzygous i nsertions HETEROzygous i nsertions that i nsert AA HETEROzygous i nsertions that cause frameshifts i n genes HETEROzygous i nsertions within genic regions skewed HETEROzygous i nsertions skewed HETEROzygous i nsertions that i nsert AA skewed HETEROzygous i nsertions that cause frameshifts i n genes skewed HETEROzygous i nsertions within genic regions HOMOzygous fixed alternate allele compared to reference HOMOzygous Osynonymous AA change HOMOzygous synonymous AA change HOMOzygous fixed alternate allele compared to reference within genic region HOMOzygous replace stop codon with new stop codon HETEROzygous replaces stop codon with new stop codon HOMOzygous stop to readthrough HETEROzygous stop to readthrough HOMOzygous creates premature stop codon HETEROzygous creates premature stop codon HETEROzygous Replaces stop codon with new stop codon skewed freq HETEROzygous stop to readthrough skewed freq HETEROzygous creates premature stop codon skewed freq At l east 2 alternate alleles but the alternate alleles at skewed freq (<=0.YY) l ower quality SP but probably real skewed freq SP creates Osynonymous AA mutation skewed freq SP creates synonymous AA mutation Skewed freq SP within genic region High quality SP synonymous AA mutation High quality SP Osynonymous AA mutation High quality SP genic region High quality SP High quality HOMOzygous High quality HETEROzygous G D A R SVF B/R ew Traveler Design D %.3% Priority ,95,55 5,966,36-00,772,0,27, ,9 23, ,92 559,2 Bar Emulation EXT % 77 5,59,996,5, ,377 72,23 G A R Traveler % 2 2,67,20 3,067,2 3,97 3,753 65,2 63, ,9 557,95 665,627 High Valley C6 J L B Ambush Exacto %.62% ,3,072 27,222,7 2,76,95 3,299,72 29,22 22,29 295,925 57,927 5,7,72 39, ,2 3, ,669 3,7,67 75, , ,26 7,57 2 3, ,572 56, ,052, ,6 9, 75,2 2, ,955 5,23 5,206 35, ,, ,59 37, ,6,0,7, ,29 7,52 55,5 5, ,22 29, ,76,0,0,267 6, ,6 9,36 62,7 5, ,600 97,6 3 2,0 5,77 950, 6, ,66,97 6,72 59, ,3 95, ,27 56, , , , , ,5 5, ,73 3,59,65 29, ,692 7, ,325 7, ,32 0, ,3 7, ,06, ,267 30,572, ,752 32,7 9,909,57,26 6,56, , , ,5 20 5, 202 2, , ,5 30, , ,5,272 2,35,93,72, ,777 3,295,9 20,35 9,303 9,262 72,399,529,59 2,305,606 3,3,995 Circle A 2000 Plus % ,73,932 3,726,63, , , 57,39 25, ,626,303,527,292,00 2,0,637, ,2, ,2,773,5,96 5,659 53,376 6,953 9, ,0 5,3 3,72 20, ,2 5,3 9,269 7, ,2 2, ,270 7,3 32,27 2,0 3 7,25 60, ,906,52,997 29,309 2,33,70,0,57,57,57,55,53,223,502,6 7,67 7,23 7,32 7,0 7,06 9,2,72 9,05,73,7 699, , , , , ,302,0,57 97,2,902,39 2,06,37 5,079,69 2,57 2,6 6,057,962 3,927 6,527 6,9,9 270, ,76 379,32 7,3 27,,26 6,236 7,556 9,96 9,937,77 7,72,505 9,92,2 5, 63,6 67,92 906,02 6,63,7,65,726,37,693,350 2,6,55 2,32,62 2,579,35 2,2,6 2,36,35 2,323,699 2,296,37 3,9,37,22,533,0,33 6,36,7 6,75, libraries per HiSeq 2500 lane 2 x 0 bp paired-end reads ~32X coverage $,650 o. LOF Mutations Total Genes Trim/Filter reads: Illumina adapter sequences Base quality scores Remove reads containing repetitive elements Error correct using MSR-CA: Align to UMD3. using extgee: High stringency/paired-end alignment algorithm Generate mutation report Capture 0 pieces info including gene annotation Upload to PostgreSQL relational database o. Genes LOF Homozygotes o. Genes LOF Heterozygotes o. Candidate LOF Genes Removes most sequencing errors 3 umbers of Broken Genes Data Analysis We have sequenced each of these bulls to an average depth of 30X and have generated whole genome polymorphism reports... Candidates for embryonic or early developmental lethals Process mutation report in db into 65 categories (increases monthly): 263 genes with LOF alleles Some are assembly errors

7 Comparison to Human Description Avg per bull 00 Genomes Splice site +/- 2bp 5.5 UTR 25,9.3 Indels non-genic 20,239.2 Indels genic 9,72.7 Indels (inframe) that affect -2-3 amino acids (AA) Indels that cause frameshifts Stop codon usage 0.5 High quality SP synonymous AA,7. -2,000 High quality SP nonsynonymous AA, ,000 High quality SP genic region,22,60.6 High quality SP 3,52,59.0 High quality homozygous SP (differing to Dominette) 2,06,79.5 High quality heterozygous SP 5,502,7. Broken Genes Mutant On average a bull would carry 3 lethals We suspect that many are not real assembly errors Our analysis pipeline is continually evolving Many may not be essential for life We know from human studies that every human carries about 7 lethals Cows are going to be very similar We just didn t sequence enough bulls to find homozygotes that would eliminate these genes from consideration as being essential for life There still must be a LOT of broken genes floating around in the Angus population and they really must have negative effects on SOME traits Of the,77 genes with predicted loss of function alleles, 76 (.7%) are identified in the mouse genome informatics database data base as possessing a lethal phenotype (2,3 mouse genes have mouse phenotype terms which include lethal ) in knockout homozygotes They can t all be lethal. Can they?? 37 3 Objectives & Updates Sequence the genomes of additional 9 registered Angus bulls To find as many LOF alleles as possible and remove from consideration those that are found as homozygotes in any bull (can t be essential for life) Sequencing costs have decreased from $5,500 to $,650 American Angus Association $50,500 American Hereford Association $25,000 Beefmaster Breeders United $23,250 American Gelbvieh $23,250 American International Charolais, American Simmental Association, American Maine-Anjou Association, American Wagyu Association? We will sequence at least 50 bulls from 6- breeds Exchange sequence data USDA MARC, Genome Canada and IRA projects Develop a genotyping assay with: ) All LOF alleles, 2),000 SPs from BovineSP50, and 3) As many other functional variants as we can fit AFFYMETRIX will build custom assay with 53,000 SPs for ~$6 eed,000 from the BovineSP50 to impute genotypes up to 50K to allow use of developed molecular EPD (MEPD) equations Likely to find 5-6,000 LOF alleles in all breeds of cattle Remainder will be variants that are likely to have functional effect i.e., change the amino acid composition of proteins Genotype,000 heifers Missouri Show-Me-Select TM Replacement Heifer Program Recruited from multiple breeds (primarily Angus) Objectives & Updates Any LOF allele that never turns up in homozygous form is probably lethal Develop MEPDs for fertility in males and females (via embryonic loss) 39 0 Objectives & Updates Develop economic selection indexes that support multi-trait selection, inclusive of fertility Develop decision support software to optimize breeding schemes via the implementation of selection indexes and mate selection based upon sire and dam recessive lethal genotypes We will have MEPDs for about traits including Feed Intake, Shear Force, Fertility, Carcass, Growth, etc (From previously funded grants) We will also have genotypes on heifers and bulls for lethals Develop: Web-based educational training program for extension livestock specialists, allied industries, producers, veterinarians, and students to enable successful adoption of genomic technologies for beef cattle breeding decision-making Develop simulation exercise that demonstrates effects of MEPDs for heifer and sire fertility on reproductive performance and profitability Develop innovative educational and outreach materials for the enhancement of the Beef Cattle Community of Practice ADDITIVE 2 7

8 Thank you 3

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