The hunt for a functional mutation affecting conformation and calving traits on chromosome 18 in Holstein cattle
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This document discusses the research on chromosome 18 in Holstein cattle, focusing on the functional mutations affecting conformation and calving traits. The study employs next-generation sequencing to identify causal variants related to traits such as dystocia and calving ease. Findings suggest that structural variants in the siglec-12 gene are linked to differences in sire calving ease, highlighting the complexity of the genetic influences on these traits.
The hunt for a functional mutation affecting conformation and calving traits on chromosome 18 in Holstein cattle
- 1. The hunt for a functional mutation affecting conformation and calving traits on chromosome 18 in Holstein cattle JJ..BB.. CCoollee,,11,,** JJ..LL.. HHuuttcchhiissoonn,,11 DD..JJ.. NNuullll,,11 PP..MM.. VVaannRRaaddeenn,,11 GG..EE.. LLiiuu,,11 SS..GG.. SScchhrrooeeddeerr,,11 TT..PP.. SSmmiitthh,,22 TT..SS.. SSoonnsstteeggaarrdd,,11 CC..PP.. VVaann TTaasssseellll,,11 aanndd DD..MM.. BBiicckkhhaarrtt11 1Animal Genomics & Improvement Laboratory and 2US Meat Animal Research Center Agricultural Research Service, USDA 1Beltsville, MD and 2Clay Center, NE john.cole@ars.usda.gov 2014
- 2. Overview What do we know about chromosome 18? How can sequencing help us learn more? What did we learn when we looked at the data? How did we approach these new challenges? Where are we now? Source: Ianuzzi (Chromosome Res., 4:448–456) 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (2) Cole et al.
- 3. Introduction Several studies (Kuhn et al., 2003; Cole et al., 2009; Seidenspinner et al., 2009) have reported QTL on BTA 18 associated with dystocia Bioinformatic analysis using SNP data has not identified the causal variant Next generation sequencing (NGS) has recently been used to find causal variants for novel recessive disorders 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (3) Cole et al.
- 4. Chromosome 18 is different Markers on chromosome 18 have large effects on several traits: Dystocia and stillbirth: sire and daughter calving ease and sire stillbirth Conformation: rump width, stature, strength, and body depth Efficiency: longevity and net merit Large calves contribute to reduced cow lifetimes and decreased profitability 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (4) Cole et al.
- 5. Marker effects for dystocia complex AR-BFG-`GS-109285 ARS-BFGL-NGS-109285 Cole et al., 2009 (J. Dairy Sci. 92:2931–2946) Source: https://www.cdcb.us/Report_Data/Marker_Effects/marker_effects.cfm?Breed=HO 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (5) Cole et al.
- 6. Correlations in dystocia complex 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (6) Cole et al.
- 7. The QTL also affects gestation length Maltecca et al., 2011 (Animal Genet. 42:585-591) 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (7) Cole et al.
- 8. The dystocia complex The key marker is ARS-BFGL-NGS-109285 at (rs109478645 ) 57,589,121 Mb on BTA18 Intronic to Siglec-12 (sialic acid binding Ig-like lectin 12) Recent results indicate effects on gestation length (Maltecca et al., 2011) and calf birth weight (Cole et al., 2014), as well as calving traits (Purfield et al., 2014) 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (8) Cole et al.
- 9. Where did it come from? Source: http://bit.ly/VsIups Source: https://www.cdcb.us/CF-queries/Bull_Chromosomal_EBV/bull_chromosomal_ebv.cfm? 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (9) Cole et al.
- 10. Who popularized it? 57,861 daughters 2 million granddaus Source: http://bit.ly/1BkTTsE. Maternal haplotype from Ivanhoe Source: https://www.cdcb.us/CF-queries/Bull_Chromosomal_EBV/bull_chromosomal_ebv.cfm? 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (10) Cole et al.
- 11. This is a gene-rich region Discussed on Tuesday (Abstract 288, Mao). http://useast.ensembl.org/Bos_taurus/Location/View?r=18%3A57583000-57587000 http://www.ncbi.nlm.nih.gov/gene?cmd=Retrievedopt=Graphicslist_uids=618463 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (11) Cole et al.
- 12. Copy number variants are present Hou et al. 2011 (BMC Genomics,12:127) ARS-BFGL-NGS-109285 is flanked by CNV There’s a loss and a gain to the left (8 SNP region) There’s a gain to the right (10 SNP region) This can result in assembly problems 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (12) Cole et al.
- 13. What if we look at a different trait? Cole et al. (2009) proposed the following mechanism: Siglec-12 may sequester circulating leptin This increases gestation length Calf birth weight (BW) is higher because of increased gestation length Higher BW is associated with dystocia 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (13) Cole et al.
- 14. We don’t have birth weight data Birth weights are not routinely recorded in the US Collaborated with Hermann Swalve’s group to develop a selection index prediction of BW PTA Performed GWAS and gene set enrichment analysis to search for interesting associations (Cole et al., 2014, JDS 97:3156-3172) 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (14) Cole et al.
- 15. GWAS for birth weight PTA h Cole et al., 2014 (J. Dairy Sci., 97:3156–3172) 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (15) Cole et al.
- 16. Are we measuring anything new? Identified a SNP on BTA16 intronic to LHX4, which is associated with cow body weight and length (Ren et al., 2010, Mol. Bio. Reprod., 37:417-422). 4 SNP in the QTL region on BTA 18 had large effects Several other SNP with large effects intronic or adjacent to genes with unknown functions 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (16) Cole et al.
- 17. KEGG pathways for birth weight What does regulation of the actin cytoskeleton have to do with birth weight in cattle? That is, do these results make sense? Maybe…these pathways may be involved in establishment maintenance of pregnancy, as well as coordination of growth and development. Cole et al. (2014) 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (17) Cole et al.
- 18. Pedigree haplotype design Arlinda Chief AA, SCE: 8 Chief AA, SCE: 7 MGS Arlinda Rotate AA, SCE: 8 δ = 10 Tradition Melwood Aa, SCE: 8 CMV Mica Aa, SCE: 14 Jed Aa, SCE: 15 Leduc Aa, SCE: 18 Aa, SCE: 10 MGS These bulls carry the haplotype with the largest, negative effect on SCE: Rockman Ivanhoe Aa, SCE: 6 Delegate Aa, SCE: 15 Laramie aa, SCE: 15 Couldn’t obtain DNA: Combination ??, SCE: 7 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (18) Cole et al.
- 19. How many scientists does it take… You just missed his talk (Abstract 164, Bickhart et al.)! You went to her poster on Tuesday (Abstract 799, Cooper et al.), right? He’s back in Maryland, working. 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (19) Cole et al.
- 20. Sequencing coverage Bull name SCE1 Genotype2 Total reads Coverage Pawnee Farm Arlinda Chief 7 AA 333,628,731 12.03 Glendell Arlinda Chief 8 AA 981,726,824 35.41 Sweet Haven Tradition 10 Aa 390,387,538 14.01 Arlinda Rotate 8 AA ~476,000,000 17.00 Arlinda Melwood 8 Aa ~448,000,000 16.00 Juniper Rotate Jed 15 Aa 656,190,604 23.66 CMV Mica 14 Aa 433,353,161 15.63 Lystel Leduc 18 Aa 767,440,677 27.68 Willow-Farm Rockman Ivanhoe 6 Aa 195,769,690 7.06 Cass-River Select Delegate 15 Aa 377,380,110 13.61 Wedgwood Laramie 15 aa 371,477,172 13.39 1Predicted transmitting ability (PTA) for sire calving ease, the percentage of offspring born with difficulty. Small values are desirable and large values are undesirable. 2The genotype of the tag SNP for the QTL, where “A” and “a” are the major and minor alleles, respectively. 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (20) Cole et al.
- 21. Results from Illumina sequencing Data analyzed using paired-end read alignments and split-read mapping Portions of two exons and a connecting intron within the Ig-like protein domains may have been duplicated Some heterozygotes with desirable SCE also have deletions near the N-terminal end of the protein 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (21) Cole et al.
- 22. Possible assembly problem on BTA18 This could be a GC-rich region (bias in Illumina chemistry). More reads than expected may align here because repetitive elements were combined during assembly. 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (22) Cole et al.
- 23. Genome assembly (simplified) Reads must be assembled into chromosomes Assembly is a computational process (Liu et al., 2009; Zimin et al., 2009) This process is imperfect – repetitive regions are hard to assemble correctly! Sometimes, this… should be this. 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (23) Cole et al.
- 24. Can it be corrected using long reads? BTA18 genomic DNA extracted from CHORI-240 BAC library (L1 Domino 99375) at AGIL Source: Pacific Biosystems Sequencing libraries constructed at USDA MARC, pooled, and run on PacBio RS II BAC ID Insert size (bp) Start End CH240-389P14 174,682 56,954,654 57,129,335 CH240-234E12 178,618 57,058,248 57,236,865 CH240-280L6 175,831 57,092,237 57,268,067 CH240-34N7 158,841 57,129,383 57,288,223 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (24) Cole et al.
- 25. Processing of PacBio reads BAC DNA was pooled at MARC to have enough material to construct a sequencing library Reads were assembled into contigs using HGAP in SMRTanalysis v2.2.0 44 contigs with an N50 of 31 kb were constructed 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (25) Cole et al.
- 26. Analysis of alignments PacBio contigs aligned against UMD3.1 contigs using MUMmer 3.0 Short (Illumina) reads aligned against PacBio contigs using BWA 0.7.5a-r405 Paired-end discordancy interrogated using custom scripts (Bickhart, unpublished data) 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (26) Cole et al.
- 27. Alignment of BAC contigs with UMD3.1 A line with a slope of 1 indicates that a segment is conserved between the two sequences – this contig is almost identical between our PacBio assembly and the UMD3.1 reference assembly. 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (27) Cole et al.
- 28. Discordancy analysis Illumina reads aligned w/PacBio contigs Reads with lengths ±4σ were counted Discordancies may indicate Problems in the PacBio assembly The presence of repetitive elements Structural differences between the Holstein and Hereford (unlikely) 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (28) Cole et al.
- 29. DNA in PacBio and not in UMD3.1 20000 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 Reads map to PacBio and UMD3.1 contigs. Vector DNA – nothing to see here! ~10 kbp of DNA in PacBio contig that doesn’t map to UMD3.1! Reads map to PacBio and UMD3.1— ARS-BFGL-NGS-109285 is placed here. 0 50000 100000 150000 200000 250000 300000 scf7180000000136|quiver REF 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (29) Cole et al.
- 30. There are clearly assembly problems 25000 20000 15000 PacBio sequence duplicated 10000 5000 0 PacBio sequence duplicated on UMD3.1 contig on UMD3.1 contig 0 20000 40000 60000 80000 100000 120000 scf7180000000103|quiver REF 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (30) Cole et al.
- 31. What have we learned? This is more complex than SNP genotyping, and unsuccessful experiments are expected You needs lots of high-quality DNA for constructing PacBio libraries Overlapping BACs should not be pooled (some people already know this) Data editing and error-correction are critical 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (31) Cole et al.
- 32. Next steps Re-assemble raw reads following more stringent edits and data cleaning Re-sequence single BACs or pooled, non-overlapping BACs Sequence the RPCI-42 Holstein BACs (Monsanto calf) Are structural differences between Holstein and Angus in this region 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (32) Cole et al.
- 33. Conclusions Structural variants in and around the Siglec-12 gene are associated with differences in SCE SNP are misplaced on the UMD3.1 assembly A region ~8 kb downstream of ARS-BFGL-NGS- 109285 appears to be misassembled The causal variant on BTA18 has not yet been conclusively identified 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (33) Cole et al.
- 34. Acknowledgments Reuben Anderson and Alexandre Dimitchev, AGIL, ARS, USDA Renee Godtel, US Meat Animal Research Center, ARS, USDA USDA-ARS appropriated projects 1245-31000- 101-00 (DMB, JBC, JLH, DJN, PMV), 1245- 31000-104-00 (GEL, SGS, TSS, CPV), and 5438- 31320-012-00 (TPS) Cooperative Dairy DNA Repository and Council on Dairy Cattle Breeding 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (34) Cole et al.
- 35. Questions? 10th World Congress on Genetics Applied to Livestock Production, Vancouver, BC, Canada 21 August 2014 (35) Cole et al.
Editor's Notes
- #25: Transition/explain why PacBio & Illumina are different