CARMO, A. S. do

CHUD, T. C. S.

MACHADO, M. A.

OLIVEIRA JÚNIOR, G. A. de

PANETTO, J. C. do C.

SILVA, M. V. G. B.

VERNEQUE, R. da S.

English

Repository Open Access to Scientific Information from Embrapa

52a Reunião Anual da SociedadeBrasileira de ZootecniaZootecnia: Otimizando Recursos ePotencialidadesBelo Horizonte – MG, 19 a 23 de Julho de2015Genome Wide CNVs analysis to identify variants associated with coat color in Gyr breed1Adriana Santana do Carmo2, Gerson A. de Oliveira Júnior3, Tatiane Cristina Seleguim Chud4, João Cláudio do CarmoPanetto5, Rui da Silva Verneque6, Marco Antônio Machado7 e Marcos Vinicius Gualberto Barbosa da Silva81Parte do pós doutorado da primeira autora, financiado pelo CNPq (Processo: 407246/2013-4)2 Pós doutoranda da Embrapa Gado de Leite, Juiz de Fora, MG, Brasil. Bolsista PDJ CNPq (Processo: 150990/2014-6)e-mail: adrianasantanacarmo@gmail.com3Aluno de doutorado em Melhoramento Animal  e Biotecnologia  –  FZEA /  USP,  Pirassununga,  SP, Brasil.  email:gersonjr@gmail.com4 Aluna de doutorado em Genética Animal – Unesp / FCAV, Jaboticabal, SP, Brasil. email:tatischud@gmail.com5 Pesquisador  - Embrapa Gado del Leite, Juiz de Fora, MG, Brasil. e-mail:joao.panetto@embrapa.br6 Pesquisador  - Embrapa Gado del Leite, Juiz de Fora, MG, Brasil. e-mail:rui.verneque@gmail.com7 Pesquisador  - Embrapa Gado del Leite, Juiz de Fora, MG, Brasil. e-mail:marco.machado@embrapa.br8 Pesquisador  - Embrapa Gado del Leite, Juiz de Fora, MG, Brasil. e-mail:marcos.vb.silva@embrapa.brResumo: Diversos estudos reportaram CNVs envolvidos com a cor da pelagem em bovinos taurinos, mas um númeromuito pequeno deles explora o papel dessas variantes estruturais desse fenótipo em animais zebuínos.  Sendo assim, opresente estudo objetivou identificar os CNVs associados  aos  padrões de cor da pelagem mais comuns em animais daraça Gir. Um estudo de associação ampla do genoma do tipo caso/contole foi realizado utilizando CNVs identificadosem 445 touros, com o intuito de verificar aqueles associados a quatro contrastes entre diferentes pelagens:  Chita xChitado,  Chita  x  Monocromo,  Gargantilha  x  Monocromo e  Mouro  x  Monocromo.  Foram identificados  60  CNVssignificativamente associados e dois importantes genes candidatos (KIT e  TYRO3), além de  um QTL para a cor depelagem,  previamente  reportados  em  animais  taurinos.  Os  resultados  do  estudo  sugerem  que  as  mesmas  regiõesgenômicas podem ser responsáveis por controlar a cor da pelagem em bovinos taurinos e zebuinos. Palavras–chave:  bos taurus indicus, gwas, KIT, TYRO3, variantes estruturais, tolerância ao calorAbstract: There are several studies reporting CNVs involved with cattle coat color, but a very few number of themexplore structural variants role in indicine cattle coat color pattern. Therefore, the present study aim to identify CNVsassociated with the most commons patterns of coat color in Gyr animals.  A case/control genome wide CNV analysiswere performed on 445 Gyr bulls to identify CNVs associated with four different Gyr coat color contrasts: Chita xChitado, Chita x Monochrome, Gargantilha x Monochromes and Mouro x Monochromes.  Sixty CNV were  significantassociated among all contrasts. Two coat color important candidate genes (KIT and TYRO3) and two QTLs previouslyreported in taurine animals, overlapped with these CNVs. The study results suggests that the same genomic regions canbe  responsible to control coat color phenotype in taurine and indicine cattle. Key word: bos taurus indicus, gwas, KIT, TYRO3, structural variants, heat toleranceIntroductionIn early domesticated stocks, coat color was a variable trait.  As one of the most important features related toanimals visual identity, coat color became a trait under intense selection, which resulted in color patterns within breeds.Furthermore,  coat  color  shows  economic  significance  due  to  its  potentially  deleterious  pleiotropic  effects,  andpopularity differences, since some colors are more popular in certain parts of the world, attaining higher commercialprices . According to Smith (1996), pigmentation in cattle is determined by the presence of functional melanocytes.Abnormal migration may be one of the reasons for piebaldism phenotype, which is the mixture of pigmented andunpigmented coat areas. Among taurine breeds, this phenotype is particularly common in Holstein and Simmental andhas attracted the attention of scientific community because it is expected that animals with higher proportion of whitecoat  absorb  less  solar  radiation  and  are  therefore  better  buffered  against  heat  stress  in  tropical  and  subtropicalenvironments.   Several studies already have identified copy number variants involved with coat color in livestock.Copy number variants found on AGOUTI  gene that causes white coat color in sheep (Bickhart and Liu, 2014), and KIT________________________________________________________________________________________________________________________________________________  Página 1 de 352a Reunião Anual da SociedadeBrasileira de ZootecniaZootecnia: Otimizando Recursos ePotencialidadesBelo Horizonte – MG, 19 a 23 de Julho de2015gene, which is involved with coat spotting in several cattle breeds, have been considered as candidates to understandcoat color expression (Reinsch et al., 1999). Nowadays, few studies have been conducted in order to explore the structural variants responsible for thisphenotype in indicine cattle, thus the present study aim to identify CNVs associated with the most common patterns ofcoat color in Gyr animals. Material and MethodsA total of 445 Gyr bulls were genotyped using the Illumina BovineHD Genotyping BeadChip (Illumina Inc.,San  Diego,  CA).  Animals  were  classified  according  to  their  coat  color  patterns  into  five  groups:  Chitado,  Chita,Gargantilha, Monochrome and Mouro. The Gyr coat color were used as qualitative phenotype for genome-wide study.The Log R Ratios (LRR) from 777,961 SNP probes generated by BovineHD Genotyping BeadChip were imported fromthe GenomeStudio software into Golden Helix SNP & Variation Suite (SVS) 8.3.0 (Golden Helix Inc., Bozeman, MT,USA) and a total of 695,818 SNPs were mapped onto the Bos taurus genome assembly UMD 3.1 within 29 autosomeschromosomes.Previously to CNVs identification, a waviness effect correction were applied to LRR based on genome GCcontent. The copy number analysis module (CNAM) under the multivariate option was used to segment chromosomeswith  a  maximum of  100  segments  per  window (10,000  markers),  a  minimum of  5  markers  per  segment,  and  asignificance level of p = 0.01 for pairwise permutations (n = 1,000) as described by Xu et al., (2014). A case/control study was performed to test the following contrasts between coat color patterns: A - Chita xChitado (n = 75 x  249),  B - Chita x Monochrome (n = 75 x 47), C - Gargantilha x Monochromes (n = 51 x 47) and D -Mouro x Monochromes (n = 22 x 47). A linear regression under the additive genetic model was employed to identifyCNVs associated with each contrast. Significant CNVs were counted at FDR significance level of (p-value) < 0.05.The database Variation gene 79 from  Biomart (Ensembl)  and cattle QTLdb were used to map significantCNVs overlapping  genes and previously reported QTLs  to functionally explore candidates genomic regions.Results and DiscussionFrom 1461 unique CNVs detected, a total of 60 CNVs were significant considering all contrasts (Table1).  Thecontrast D presented the higher number of significant CNVs (n = 51), followed by contrasts A (n = 7) and B (n = 2). Thecontrast C did not show any significant CNV.  Gene content analysis demonstrated that  CNVs overlap 734 genes,highlighting  stem cell growth factor receptor c-Kit (KIT) and tyrosine-protein kinase receptor (TYRO3). These geneshave already been reported as important candidates involved  with coat color in taurine breeds.  The KIT gene is included in a region denominated  spotting locus mapped to bovine chromosome 6 (BTA6)(Grosz and MacNeil, 1999). In the same chromosome region, Reinsch et al. (1999) and Liu et al.(2009) localised a QTLfor the degree of spotting in German Holstein-Friesian and Simmental cattle and in a Holstein-Friesian x Jersey cross.Besides cattle, kit mutation was described as responsible for pigmentation defects in human, mouse and horse  (Geissleret al., 1988; Giebel and Spritz,  1991; Haase et al., 2007).  According to Haltaufderhide and Oancea (2014), both KITand TYRO3 are among the 10 highest expressed receptor kinase genes in human epidermal melanocytes. Durkin et al. (2012) reported that color sidedness in Belgian Blue cattle is determined by a first allele onchromosome 29 (Cs (29)), which results from the translocation of a 492 kb chromosome 6 segment encompassing KITto chromosome 29. Although, our study were able to find a significant CNV located on chromosome 29, the duplicationwas found in BTA29:31934125 (first  SNP position),  which is approximately 10 Mb base pairs upstream from thetranslocation described by the authors (BTA29:20.8 – 21.2Mb). The QTL content analysis demonstrated CNVs overlapping a QTL that were previously associated with coatcolor (QTL ID = 6270) in taurine breeds, which reinforce the potential importance of these region as a candidate. Thesignificant CNvs also overlaps three QTLs involved with heat intensity (QTL IDs = 3569, 5016, 5004) and one QTLinvolved with heat stress (QTL IDs = 31192). These findings agree with Mader et al. (2002) that demonstrated that coatcolor had a large impact on the heat tolerance of cattle.ConclusionsResults suggests that the genomic regions controlling coat color phenotype in both bovine subspecies (taurineand indicine) are similar, and they are associated to coat color in Gyr cattle which are also involved with heat tolerancecontrol. ________________________________________________________________________________________________________________________________________________  Página 2 de 352a Reunião Anual da SociedadeBrasileira de ZootecniaZootecnia: Otimizando Recursos ePotencialidadesBelo Horizonte – MG, 19 a 23 de Julho de2015Table 1. CNVs associated with color coat contrasts in Gyr Breed.Coat color contrasts CNV predictor aChita x ChitadoBovineHD0200033194, BovineHD0300030854, BovineHD0800004068,BovineHD1100031344, BovineHD1200009631, BovineHD1600013398,BovineHD2000000005Chita x Monochrome BovineHD0600019880, BovineHD1400008618Mouro x MonochromesBTB-00199499, BovineHD0100006355, BovineHD0100018402,BovineHD0100018845, BovineHD0100029600, BovineHD0100047216BovineHD0200007767, BovineHD0200028760, BovineHD0200038484,BovineHD0300004515, BovineHD0400026873, BovineHD0400032672BovineHD0500011305, BovineHD0500019746, BovineHD0500021645,BovineHD0600005556, BovineHD0600026173, BovineHD0700019948BovineHD0700033340, BovineHD0800007537, BovineHD0800011514,BovineHD0800018380, BovineHD0900009034, BovineHD0900020346BovineHD0900025424, BovineHD1000010927, BovineHD1000019695,BovineHD1100006145, BovineHD1300006924,BovineHD1300006985BovineHD1400010215, BovineHD1500000585, BovineHD1500013027BovineHD1500022519, BovineHD1600006751, BovineHD1600016891BovineHD1700002291, BovineHD1700002359, BovineHD1800005170BovineHD1800005366, BovineHD1800007727, BovineHD2000005489BovineHD2000013128, BovineHD2000013512, BovineHD2100017701BovineHD2200006503, BovineHD2300014492, BovineHD2400012148BovineHD2700000001, BovineHD2900009553, Hapmap23994-BTA-141224a first SNP from CNV ReferencesBickhart,  D.  M.;  Liu,  G.E.  The  challenges  and  importance  of  structural  variation  detection  inlivestock. Frontiers in Genetics. p. 5:37. doi:10.3389/fgene.2014.00037, 2014.Durkin,  K.;  Coppieters,  W.;  Drögemüller, C. et  al.  Serial  translocation by means of  circular intermediatesunderlies color sidedness in cattle. Nature, v. 482, p. 81–84. doi: 10.1038/nature10757, 2012.Geissler, E. N.; Ryan, M. A.; Housman, D. E. The dominant-white spotting (W) locus of the mouse encodesthe c-kit proto-oncogene. Cell, v.55, p.185-192. 1988.Giebel, L. B.; Spritz, R. A. Mutation of the KIT (mast/stem cell growth factor receptor) protooncogene inhuman piebaldism. Proc. Natl. Acad. Sci. USA 88:8696-8699, 1991.Haase, B.; Brooks, S. A.; Schlumbaum, A. et al. Allelic heterogeneity at the equine KIT locus in dominantwhite (W) horses. PLoS Genet. 3:e195., 2007.Haltaufderhyde, K. D.; Oancea. E. Genome-wide transcriptome analysis of human epidermal melanocytes.Genomics, v. 104, p. 482-489, 2014.Liu, L.; Harris, B.; Keehan, M.; Zhang, Y. Genome scan for the degree of white spotting in dairy cattle. Anim.Genet. v. 40, p. 975-977, 2009.Mader, T.L., S.M. Holt, G.L. Hahn, M.S. Davis, and D.E. Spiers. 2002. Feeding strategies for managing heatload in feedlot cattle. J. Anim. Sci. 80: 2373- 2382.Reinsch, N.; Thomsen, H.; Xu, N.; Brink, M. et al.A QTL for the degree of spotting in cattle shows syntenywith the KIT locus on chromosome 6. J. Hered. v. 90, p.629-634, 1999.Smith, B. P. Large animal internal medicine:diseases of horses, cattle, sheep and goats, 2nd edition. St Louis,Mosby, 1996.Xu, L.; Hou, Y,.; Bickhart, D. M. et al. A genome-wide survey reveals a deletion polymorphism associatedwith resistance to gastrointestinal nematodes in Angus cattle. Funct Integr Genomics. v. 14, p.333-339, 2014. ________________________________________________________________________________________________________________________________________________  Página 3 de 3

Genome Wide CNVs analysis to identify variants associated with coat color in Gyr breed.

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Genome Wide CNVs analysis to identify variants associated with coat color in Gyr breed.

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