University of Illinois at Urbana-Champaign Department of Animal Sciences Identification of a nonsense mutation in APAF1 that is causal for a decrease in.

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University of Illinois at Urbana-Champaign Department of Animal Sciences Identification of a nonsense mutation in APAF1 that is causal for a decrease in reproductive efficiency in dairy cattle H. A. Adams 1,2, T.S. Sonstegard 3, P. M. VanRaden 4, D. J. Null 4, C.P. Van Tassell 3, D.M. Larkin 1, and H. A. Lewin 1,2,5 1 Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA, 2 Institute of Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA, 3 Bovine Functional Genomics Laboratory, Agricultural Research Service, USDA, Beltsville, MD, USA, 4 Animal Improvement Programs Laboratory, Agricultural Research Service, USDA, Beltsville, MD, USA, 5 Department of Evolution and Ecology, University of California, Davis, Davis CA 95616, USA With the recent advent of genomic tools for cattle, several mutations affecting fertility have been identified and selected against, such as AM, PHA, DUMPS, CVM and BLAD. In an attempt to identify unknown recessive lethal alleles in the current dairy population, a search using deep Mendelian sampling of more than 100,000 Holstein cattle was conducted for high-frequency haplotypes that have a deficit of homozygotes at the population level. This search led to the discovery of a putative recessive lethal in Holstein dairy cattle on BTA5. The haplotype denoted HH1 was associated with reduced fertility, and further investigation identified one highly-influential Holstein bull as the source ancestor. By combining whole-genome resequencing, high-density genotyping and haplotyping techniques, a nonsense mutation in APAF1 was identified, which is predicted to truncate approximately one-third of the encoded APAF1 protein. No homozygous recessive individuals were found in 758 genotyped animals, whereas all known carriers and carrier haplotypes possessed one copy of the mutant allele. Functional annotation of APAF1 supports our findings and the gene’s vital role in development. This newly identified lethal has been responsible for a substantial number of spontaneous abortions in Holstein dairy cattle throughout the past half-century. With the mutation identified, selection against the deleterious allele in breeding schemes will aid in eliminating this defect from the population. Whole-genome resequencing thus proved to be a powerful strategy to rapidly identify a deleterious mapped mutation in a known carrier of a recessive lethal allele. INTRODUCTION P VanRaden, P.M., Null, D.J. Olson, K.M., & Hutchison, J.L. J. Dairy Sci. 94(12), (2011). 2.VanRaden, P.M., O’Connell, J.R., Wiggans, G.R. & Weigel, K.A. Genet. Sel. Evol. 43, 10 (2011). 3.Cecconi, F., Alvarez-Bolado, G., Meyer, B.I., Roth, K.A., & Gruss, P. Cell 94(6), (1998). 4.Muller, M., et al. Devel. Dynam. 234, (2005). 5.Yoshida, H., et al. Cell 94(6), (1998). ABSTRACT A dairy cow’s health, milk production volume and number of successful gestations are keys to maintaining a profitable dairy herd. If the reproductive performance of a cow decreases, losses are accrued in both overall production and the introduction of replacement animals into the herd. Several mutations have been identified within dairy cattle that adversely affect fertility, and selection practices targeted against transmitting the mutations to possible offspring have almost entirely eliminated the disorders from the population [1]. These lethal mutations are economically detrimental both by causing fertility loss and resulting in loss of potential replacement animals because of a lack in survival to a productive stage. In an attempt to identify unknown recessive lethals in the current dairy cattle population, a search was conducted using deep Mendelian sampling of haplotypes for which less than the expected number of homozygotes was found [2]. This search recently led to the discovery of the approximate location for several recessive lethal haplotypes in Holstein dairy cattle. One of these haplotypes targeted that contains a putative mutation, denoted HH1, has previously been associated with undesirable effects on fertility [1]. MATERIALS AND METHODS Source of targeted haplotype An initial search for lethal recessives identified HH1 as a 75-marker haplotype on cattle chromosome 5 (BTA5) spanning approximately 7 Mbp, located from coordinate 58,638,702 to 65,743,920 on the UMD 3.0. Twenty-three homozygotes were expected, none were observed, and calf losses for HH1 occurred evenly throughout gestation. The source haplotype for HH1 traced to a single source ancestor, Pawnee Farm Arlinda Chief (Chief; Table 1). Chief’s significant influence within the Holstein breed is supported by his performance records. His daughters produced > 2 liters/day more milk than daughters of other bulls born in Chief has accumulated > 16,000 daughters, > 500,000 granddaughters, and > 2 million great-granddaughters via AI. Haplotype detection and crossover analysis Fine mapping was accomplished by determining if any genotyped live animals had both the source haplotype and a crossover haplotype. Crossovers were detected using 78,465 animals genotyped for 54,001 SNPs with the Illumina BovineSNP50 BeadChips (Illumina, San Diego, CA). Frequency of HH1 heterozygotes over all animals genotyped is 3.2% for the source haplotype and 4.2% when crossover haplotypes are included. Alignment and mapping Two sons of Chief, Mark and Ivanhoe Chief, were identified as carriers of the suspect allele. An additional son of Chief, Valiant, was not heterozygous for the putative mutation, but was included in the study as a control. The genomes of Chief, Ivanhoe Chief and Valiant were sequenced using sequencing by synthesis chemistry on an Illumina HiSeq 2000 platform; Mark was sequenced using 454 Titanium technology. Sequence reads were mapped against a whole bovine genome assembly (Baylor 4.0) using SOAP2 ( Detection of SNPs and genes SNPs in the suspect region of BTA5 were identified using FreeBayes ( Putative SNPs were accepted if they fit the following criteria: > 2 reads aligning in each orientation, and minimum allele sequencing quality > 20. Functional annotation of the regional variants was done using ANNOVAR ( Validation of the mutation The HH1 haplotype was identified in the U.S. dairy population based on75 SNPs on BTA5. Genotyping was performed using the BovineSNP50 assay. 758 animals were selected for APAF1 validation genotyping using a 24 SNP multiplex panel, of which 486 were presumed carriers (presence of target haplotype). A Sequenom SNP panel was designed for the validation test. Duplicate assays were performed for 12 SNPs within and flanking the APAF1 gene in the refined HH1 interval region. RESULTS MATERIALS AND METHODS cont…. REFERENCES SUMMARY AND CONCLUSIONS Here we identify a recessive lethal mutation on the HH1 haplotype in the APAF1 gene that is presently segregating in the current Holstein dairy cattle population. With the causative mutation on HH1 now identified, selection against the deleterious allele in breeding schemes will aid in eliminating the defect from the population. The “resequencing of elite sires “approach presented here proved to be a powerful strategy for the identification of a cryptic recessive lethal in the population and should be extremely useful for the detection of other mutations affecting economically important traits of dairy cattle. Table 1. Pedigree information for 4 sequenced Holstein dairy bulls: Pawnee Farm Arlinda Chief (Chief), Milu Betty Ivanhoe Chief (Ivanhoe Chief), S-W-D Valiant (Valiant), and Walkway Chief Mark (Mark) 1 Holstein breed identification number 2 Expected Future Inbreeding (EFI) of bull’s daughters ( 3 Bull’s EFI ranking, outlining relationship of bull to the Holstein cow population ( 4 Percentage of genes in common with top currently available Holstein sires; value is an indicator of the bull’s overall contribution to the Holstein breed, from a list of the top 25 most influential bulls Fine mapping narrowed the HH1 suspect area from the 75-marker haplotype to a 39-marker window (62,435,307 to 65,597,776) on BTA5 using 8 animals that inherited both the source haplotype and a crossover haplotype. The number of crossover haplotypes that included the full 75-marker suspect area was 26. Within the suspect area of 39 markers, 51 crossovers were detected. Identification of the exact position of the defect on BTA5 was aided by whole- genome resequencing of Chief and his three influential sons (Table 2). Results of the functional analysis revealed three exonic SNPs, one of which resided in gene APAF1, upon which the suspect allele produces a stopgain mutation in its homozygous recessive state (Table 3). The original amino acid [codon] within the APAF1 protein sequence changes from a glutamine [CAA] to a stop codon [TAA], disrupting protein synthesis 43 amino acids into the 62 amino acid chain. No known mutation is observed in the bovine dbSNP for this position, therefore affirming the SNP as a previously undetected mutation within exon 11 of the APAF1 gene. Chief, Mark and Ivanhoe Chief were validated as carriers of HH1, and Valiant as a non-carrier, through resequencing, SNP detection and annotation. Among the 758 animals selected for validation genotyping, 497 were presumed carriers of the mutation. Using a 24 SNP multiplex panel, only one haplotype was associated with the stopgain. All previously identified HH1 carriers for the 75 marker interval (n=497) were identified within this haplotype group. BullID 1 Birth Year SireDamPedigree EFI 2 Rank 3 Genes Shared (%) 4 Chief Pawnee Farm Reflection Admiral (138326) Pawnee Farm Glenvue Beauty ( ) Ivanhoe Chief Chief Milu Betty Ovation Ivanhoe ( ) Valiant Chief Allied Admiral Rose Vivian ( ) Mark ChiefWalkway Matt Mamie ( ) Table 2. Sequencing statistics for Chief, Ivanhoe Chief, Valiant, and Mark 1 Based on a 2.8 Gbp estimated cattle genome size 2 Statistics generated by Illumina HiSeq 2000 sequencing 3 Statistics generated by 454 sequencing BullHigh-quality reads generated DNA sequenced Read length Expected genome coverage 1 Chief Million88 Gbp100 nt31X Ivanhoe Chief Billion150.3 Gbp100 nt50X Valiant Billion110 Gbp100 nt36X Mark Million37.9 Gbp356 nt13.5X Table 3.SNP assays used for validation, genes associated with SNPs, their corresponding allele information and locations within BTA5, and concordance of alternate alleles to carrier haplotypes *Reporting 1 assay; concordance was 100% between bi-directional assays for the same SNP loci ^Only one directional assay was functional Concordance SNP Assay*Associated GeneSNP Location UMD3.0 coordinate Ref allele Alt allele Alt allele to carrier haplotypes #1 ENSBTAG38223 (dist=216988)intergenic TC #1 ENSBTAG38223 (dist=384590)intergenic TA Not Informative #1THPS_BOVINUTR AG #1THPS_BOVINUTR AG #1MPCP_BOVINintronic TA #1MPCP_BOVINintronic TA #2^IKIP_BOVINintronic TC #1APAF1exonic/stopgain CT #1APAF1 exonic/ synonmous CT #1ENSBTAG17385 exonic/ synonmous CT #1A6QP25_BOVINdownstream CT #2^ A6QP25_BOVIN (dist=117786)intergenic CT0.359 Figure 1. Concordance of genotype to HH1 status vs. genome position This work was supported by USDA, ARS CRIS projects D and 31S. Thanks to A. Beavers, M. McClure, and Geneseek for help in processing the Sequenome validation. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. The USDA is an equal opportunity provider and employer. ACKNOWLEDGMENTS A review of APAF1 identified the gene as previously being associated with embryonic lethality in mice, where knockouts lead to lethality, and deficiencies in the gene produced embryos with several malformations [3-5]. APAF1 protein is vital in the apoptotic pathway, and deficiencies in proteins in the apoptotic pathway are associated with embryonic lethals. RESULTS cont….