2.4.2. SNP Effects Analysis

The genotypic effect was analysed for those SNPs located in intragenic or in flanking genomic regions of candidate genes, which have previously been shown to be significantly associated with a bone trait (see Table 1). SNP effects for each locus were analysed as described by Wiedemann et al. [47]. For this purpose, the actual SNP genotypes were coded as ‘0’ (AA), ‘1’ (AB), or ‘2’ (BB), with the B allele representing the minor allele. The minor allele was considered the effect allele, whereas the major allele was termed ‘other allele’. All models were computed with the R package lme4 [48].

General information for 17 loci associated with the bone breaking strengths (BBS) or bone mineral densities (BMD) of the tibiotarsus (Tib) and humerus (Hum) selected for the SNP effects analysis.

¹ GGA, Gallus gallus chromosome; ² Physical position (bp) according to the GRCg6a (galGal6) genome assembly; ³ EA, effect allele (minor allele); OA, other allele (major allele); ⁴ References from the literature suggesting an association of the gene with bone stability traits.

A linear regression model adjusted for fixed factors was applied to estimate the allele substitution effects by single marker regression (SMR):

where γijklm is the observation for a bone trait, μ is the overall mean effect, Gi is the fixed effect of generation (i = 1, 2), LLj is the fixed effect of layer line (j = 1 to 4),b1 is the regression coefficient of the SNP genotype (SNPk), Sl is the random effect of sire (l = 1 to 145), and εijklm is the residual error. Standardised allele substitution effects were calculated according to model (2) after both the dependent variable and the SNP genotypes coded as ‘0’, ‘1’, or ‘2’ were standardised to have a mean of 0 and a standard deviation of 1.

To calculate the additive and dominance effects, a dominant-recessive model (DRM) was applied considering the SNP genotype as a fixed class variable. The statistical model was as follows:

where γijklm is the observation for a bone trait, μ is the overall mean effect, Gi is the fixed effect of generation (i = 1, 2), LLj is the fixed effect of layer line (j = 1 to 4), SNPk is the fixed effect of SNP genotype (k = 1 to 3), Sl is the random effect of sire (l = 1 to 145), and εijklm is the residual error. Least squares means (LSM) for the different genotypes were estimated with the emmeans package [49]. Significant differences between LSM were tested using a t-test and adjusted by the Bonferroni method. Additive and dominance effects were estimated by contrasting the respective genotypes according to the following formulas.

where a is the additive effect, d is the dominance effect, μAA and μBB are the phenotypic mean values of the homozygous genotypes, and μAB is the phenotypic mean value of the heterozygous genotype.