Computational protocol: Evaluation of Bovine chemerin (RARRES2) Gene Variation on Beef Cattle Production Traits1

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Protocol publication

[…] Steers (n = 24) from the discovery population with extreme ADG or ADFI phenotypic and of different sire lines were selected for the SNP identification population. Twelve steers with low ADG or ADFI values and 12 steers with high ADG or ADFI phenotypes were chosen. Breeds were broadly represented in the animals with high and low phenotypes. Animals were screened and excluded for medical or health issues that may have affected either feed intake or gain phenotypes. Whole blood or buffy coats were collected from the steers for genomic DNA isolation.Primer pairs to amplify the exonic and intronic boundaries of the chemerin gene from genomic DNA were designed using Primer 3 (Rozen and Skaletsky, ). DNA sequences used as templates for primer design were obtained from the chromosome 4 Btau4.0 assembly. Oligonucleotide primers were synthesized by IDT (Integrated DNA Technologies, Coralville, IA, USA). PCR was performed in a DNA engine Dyad® peltier thermal cycler (Bio-Rad, Hercules, CA, USA). PCR reactions included 0.25 U Hot Star Taq polymerase (Qiagen, Valencia, CA, USA); 1× supplied buffer; 1.5 mM MgCl2; 80 μM dNTPs; 0.33 μM each primer; and 25 ng genomic DNA in 12 μl reactions. A portion of the PCR reaction (3 μl) was electrophoresed on 2% agarose gels to determine quality of amplification. The remainder was treated with 0.1 U exonuclease I (USB, Cleveland, OH, USA) and prepared for sequencing. Sequencing reactions were precipitated with 70% isopropanol and sequenced on an ABI 3730 sequencer (Applied Biosystems, Foster City, CA, USA). Bases were called with Phred and assembled into contigs with Phrap. Polymorphisms were identified using Polyphred and assessed using Consed. [...] The program MTDFREML (Boldman et al., ) was used to analyze markers for significant associations. Twin animals and cross-fostered animals were excluded from the analysis. The data were analyzed using an animal model that included the fixed effects of year and barn. Covariates of age and heterosis were also included. Calf and dam breed compositions were modeled with covariates for proportions Angus, Hereford, Simmental, Limousin, Charolais, Gelbvieh, Red Angus, and MARC III. Covariates for expected calf heterosis were computed from parental breed composition. SNP were fitted separately in the association model. Polygenic and breed effects were included to reduce the effects of family structure on breed- and family specific alleles (Kuehn et al., ; Goddard and Hayes, ). Variance components for polygenic effects and error were estimated using MTDFREML (Boldman et al., ). Nominal significance values were computed. Analyses of significant SNP in the validation population followed the same model definition. [...] Linkage disequilibrium (LD; r2) was defined for the 16 SNP on chromosome 4 using Haploview 4.0 software (Barrett et al., ). Blocks of LD were based on pairwise LD values. Haploview settings were as follows: the exclusion of animals with >50% missing genotypes, ignoring pairwise comparisons of markers >500 kb apart, the percentage of genotypes ≤50% and minimum minor allele frequency of 0.001. […]

Pipeline specifications

Software tools PolyPhred, MTDFREML, Haploview
Applications Sanger sequencing, GWAS
Organisms Bos taurus, Homo sapiens
Diseases Metabolic Diseases, Headache Disorders