Fine Mapping and Discovery of Recessive Mutations that Cause Abortions in Dairy Cattle P. M. VanRaden 1, D. J. Null 1 *, T.S. Sonstegard 2, H.A. Adams 3, C.P. Van Tassell 2, and K.M. Olson 4 1 Animal Improvement Programs Laboratory, ARS, USDA, Beltsville, MD 2 Bovine Functional Genomics Laboratory, ARS, USDA, Beltsville, MD 3 Institute of Genomic Biology, University of Illinois, Urbana, IL 4 National Association of Animal Breeders, Columbia, MO Abstr. LB IntroductionSequencing and Discovery of HH1 50K genotypes were used for haplotype discovery. Methods tested on Brachyspina. Numbers of expected homozygous animals were calculated using real matings and ranged from 7 to 90 for haplotypes examined further. Fertility records were used to confirm lethality, the number of carrier x carrier matings ranged from 936 to 52, haplotypes, named by breed and number within breed, (Table 1) were confirmed to be lethal with affects on conception rate (CR) ranging from 3.0% to 3.7%. CR loss occurs <60 days HH3 and JH1, <100 days for HH2, and throughout gestation for HH1 (Figure 1). Conclusions Fine Mapping the Haplotypes Objectives Locate haplotypes with significant expected homozygous animals but none observed. Narrow down suspect region using fine mapping. Use sequence data to locate the causative mutation within the fine mapped region. Explore ways to use causative mutation test results in the future. Chromo -someLocation Carrier FreqFounder NameBTAMbase%Birth year HH HH HH JH BH Table 1. Haplotypes affecting fertility. Figure 1. CR loss during gestation. Source haplotype is ~75 markers or ~ 5 Mb. Only used crossovers between a 50K animal and a 50K parent. Animals that have the source haplotype and a crossover of the source haplotype were used for fine mapping. Interval that never becomes homozygous in live animals is the suspect region. Haplotypes narrowed down to a suspect region ranging from 4.5 Mb for BH1 to 0.8 Mb for JH1 (Figure 2). Finding Haplotypes Affecting Fertility HH1 fine mapped to 3.2 Mb suspect region. Founder bull (Chief) and 3 sons (Mark, Ivanhoe Chief, and Valiant) were sequenced by U. Illinois. Sequence analysis found 12 candidate SNPs but only 3 exonic. The suspect allele for a SNP in gene APAF1 produces a stopgain mutation in its homozygous recessive state. APAF1 knockout also causes embryonic loss in mice. 758 animals, including 486 HH1 carriers, were selected for APAF1 validation genotyping. Concordance was 100% between HH1 carrier status and APAF1 mutation carrier status. Sequencing and Discovery of JH1 Source Combined With Source Suspect Area Carrier Possible Figure 2. Fine Mapping of JH1. 5 haplotypes affecting fertility have been reported to breeders since August Suspect regions narrowed using crossovers. Sequence data revealed the causative mutations for 2 of the 5, located in APAF1 for HH1 and CWC15 for JH1. Imputation can combine information from both SNPs and QTLs to determine status. Mating of heterozygotes should be avoided. Tracking Known Recessives Located the source haplotype for 3 BS and 3 HO known recessives (Table 2). RecessiveBrdBTA Tested animals Concord- ance (%) New carriers BLADHO111, DUMPSHO13, MulefootHO WeaverBS SMABS SDMBS Table 2. Using SNP haplotypes to track known recessives. Imputing Causative Mutations Carriers can be determined by haplotypes, gene tests, or combining and imputing. For JH1 730 causative mutation test results were used to impute test results for 6,784 50K animals. Causative mutation test results were also imputed for the HO known recessives CVM, BLAD and DUMPS; 84,713, 83,694 and 92,234 tests were imputed respectively. New carrier animals were discovered through imputation; 2,176 for CVM, 332 for BLAD and 3 for DUMPS. Large numbers of genotyped animals and affordable DNA sequencing now allow rapid discovery of new recessive defects. Breeders can use haplotype tests or gene tests in selection and mating programs. JH1 fine mapped to 0.8 Mb suspect region. Founder bull (Soldier) and 10 other carriers sequenced at 30X coverage by BFGL with funding from American Jersey Cattle Association. 15 candidate SNPs found but only 1 exonic. Causative mutation (stopgain) was found in CWC15 gene. 758 animals, including 486 JH1 carriers, were selected for CWC15 validation genotyping. Concordance was 99.3% between JH1 carrier status and CWC15 mutation carrier status. HH2, HH3, BH1 Sequencing These methods were automated to update the suspect region and report haplotypes monthly. Breeders receive official haplotype status for all genotyped animals (3K are unofficial). Sequenced exome (expressed DNA) for 8 carriers of each defect. Causative mutations are not obvious yet. BH1 includes 2 consecutive SNPs that never become homozygous. Some genomic evaluations may exclude these because of Hardy-Weinberg edits, but could indicate location of defect. Acknowledgments Sequencing and analysis of HH1 conducted by H. Lewin, D. Larkin, A. Beavers, M. McClure, and Geneseek. Fertility analyses conducted by J. Hutchison.