Computational protocol: A genome scan for milk production traits in dairy goats reveals two new mutations in Dgat1 reducing milk fat content

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Protocol publication

[…] For QTL detection, both linkage analyses (LA) and linkage disequilibrium (LD) using interval mapping were applied to the data using the QTLMap software (ref. ; http://dga7.jouy.inra.fr/qtlmap/). For LA, interval mapping was performed with the likelihood ratio test (LRT) using within-sire linear regression. The QTL effect (average substitution effect) was expressed in deviation units (SD). Linkage disequilibrium was based on a regression analysis of the phenotypes onto founder haplotypes. Analyses were performed for each haplotype of four consecutive SNPs along the chromosome. The computations of phase and transmission probabilities were optimized to be rapid and as exact as possible.Chromosome-wide significance levels were calculated with QTLMap, using the current family structure and the MY phenotypes. For LA, the empirical 5% and 1% chromosome-wide significance levels of the test statistics were estimated from 1,000 within-family permutations for each chromosome. For LD, the empirical chromosome-wide significance level of the test statistics was estimated from 1,000 simulations for each chromosome, assuming a trait of heritability equal to 0.35. The 5% genome-wise thresholds were obtained by applying the Bonferroni correction Pgenome-wise = 1 - (1 - Pchromosome-wise)n, where n is the number of autosomes analyzed, i.e. 29.The 95% confidence intervals of the QTL locations were estimated with the logarithm of odds drop-off implemented in QTLMap software. In practice, the bounds of the interval were the two locations where the likelihood was equal to the maximum likelihood minus 3.84 [=x(1.0.05)2]. [...] Because evidence already existed for high conservation between cattle and goat DGAT1 sequences, the 1785 bp Bos taurus mRNA sequence (NM_174693.2) was used to identify the orthologous DGAT1 region in the Capra hircus CHIR_1.0 genome and the exonic regions, through sim4 program. Using this information, N blocks (>9 consecutive undetermined nucleotides) were identified in the reference sequence in the DGAT1 orthologous region extended to 15 kb upward and forward. PCR amplifications were performed to produce DNA fragments containing one or several N blocks, using the Long PCR Enzyme Mix provided by Fermentas (http://www.fermentas.de). Either PCR or internal primers were used for the Sanger sequencing reaction with the BigDye Terminator v3.1 Cycle Sequencing Kit (http://www.appliedbiosystems.com), after ExoSAP treatment, and amplicons were analyzed on a ABI3730 DNA analyzer (ThermoFisher Scientific).Sequences were aligned using DNAbaser software (http://www.dnabaser.com/) to generate a consensus sequence from two Alpine and two Saanen animals with extreme phenotypes for fat content.SNP discovery was carried out by Sanger sequencing on the same animals. The genotypes of the discovered SNPs were determined using the Sanger sequencing method described above for the relevant PCR products of the remaining bucks of the QTL design.The primers used in this study are listed in Table . [...] The sequence of the goat DGAT1 gene was then translated and the corresponding protein sequence was compared to those of other ruminants, The sheep and bovine sequences were extracted from Uniprot (http://www.uniprot.org/ entries A8VJM4 and Q8MK44 respectively). The weblogo software (http://weblogo.threeplusone.com/) was used to obtain a graphical representation of the conservation of the protein between three domestic ruminant (cattle, sheep and goats). Predicted protein sequences of other ruminant species (Ceratotherium simum; Bulbalus bubalis; Camelus dromedaries; Camelus bactrianus and Vicugna_pacos) were extracted from NCBI (http://www.ncbi.nlm.nih.gov/gene/?term=dgat1). A percent identity matrix was then created between all the species retained using the Clustal omega software (http://www.ebi.ac.uk/Tools/msa/clustalo/). […]

Pipeline specifications

Software tools QTLMap, Sim4, WebLogo, Clustal Omega
Applications WGS analysis, Nucleotide sequence alignment, Genome data visualization
Organisms Capra hircus