| Summary: | SMAD4, SMAD5 and SMAD7 belonging to the transforming growth factor β (TGF-β) superfamily are indispensable for oocyte formation and development, ovarian organogenesis and folliculogenesis. However, only a few studies have investigated the characteristics of <i>SMAD4</i>, <i>SMAD5</i> and <i>SMAD7</i> in Tibetan sheep and the effect of their polymorphism on litter size. In this study, we examined the expression of <i>SMAD4</i>, <i>SMAD5</i> and <i>SMAD7</i> in 13 tissues of Tibetan sheep by reverse transcription-quantitative polymerase chain reaction. Further, cDNA of these genes was cloned, sequenced and subjected to bioinformatics analysis. DNA sequencing was also used to detect single nucleotide polymorphisms (SNPs). However, iM-LDR<sup>TM</sup> technology was used for SNP genotyping. Associations between polymorphisms and litter size were analyzed using data from genotyping of 433 Tibetan sheep. The results showed that the expression of <i>SMAD4</i>, <i>SMAD5</i> and <i>SMAD7</i> genes was ubiquitous in the tissues of Tibetan sheep, such as the ovary, uterus and oviduct, hypothalamus, hypophysis, heart, liver, spleen, lung, kidney, rumen, duodenum and <i>longissimus dorsi</i>. However, the expression was unbalanced and upregulated in the spleen, lung, ovary and uterus and downregulated in the <i>longissimus dorsi</i>. The bioinformatics analysis showed that <i>SMAD4</i>, <i>SMAD5</i> and <i>SMAD7</i> in Tibetan sheep encoded proteins of 533, 465 and 427 amino acids, respectively. Sequence homology analysis of the three proteins among other animals showed that the sequences of <i>SMAD4</i>, <i>SMAD5</i> and <i>SMAD7</i> of Tibetan sheep were similar to those in sheep, yak, cattle, dog, human, pig, chimpanzee, rhesus monkey and house mouse. Two synonymous mutations, g.51537A>G and g.319C>T, were detected in <i>SMAD5</i> and <i>SMAD7</i>, respectively. The associations of these SNPs and litter size were determined, and it was found that both g.51537A>G and g.319C>T have no significant effect on the litter size of Tibetan sheep. The results provided novel insights into the molecular characterization, expression profiles and polymorphisms of <i>SMAD4</i>, <i>SMAD5</i> and <i>SMAD7</i> in Tibetan sheep, but our results do not support associations between these genes and the litter size of Tibetan sheep.
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