Additional file for paper Functional validation of gene IL6R and LEPR on reproductive traits in dairy cattle

Genetic selection of milk yield traits alters the energy distribution of high producing cows, resulting in gene-induced negative energy balance, and consequently, poor body condition scores and reduced reproductive performances. Here, we investigated two metabolic-syndrome pathway genes, <em>IL6R</em> (Interleukin 6 receptor) and <em>LEPR </em>(Leptin receptor)<em>,</em> for their effects on reproductive performance in dairy cows, by applying association analyses in 1,588 Chinese Holstein cows (at a population level) and gene expression analyses in granulosa cells isolated from eight cows (at a cell level). Of the six single nucleotide polymorphisms we examined (two SNPs for <em>IL6R</em> and four SNPs for <em>LEPR</em>), five were significantly associated with at least one reproductive traits, including female fetility traits covering both the abilitiy to to recycle after calving and the abilitiy to concive and keep pregnancy when inseminated properly, and calving traits. Notably, the identified variant SNP g.80143337A/C in <em>LEPR</em> is a missense variant. The role of <em>IL6R</em> and <em>LEPR</em> in cattle reproduction were further confirmed by observed differences in relative gene expression levels amongst granulosa cells with different developmental stages. Collectively, the functional validation of <em>IL6R</em> and <em>LEPR</em> performed in this study improved our understanding of cattle reproduction while provided important molecular markers for genomic selection of reproductive traits in high-yielding dairy cattle.