Molecular Characterization, Expression Profiles of SMAD4, SMAD5 and SMAD7 Genes and Lack of Association with Litter Size in Tibetan Sheep

SIMPLE SUMMARY: Selection of molecular markers used in the marker-assisted selection of the litter size of sheep is critically essential. In this study, we identified single nucleotide polymorphisms (SNPs), g.51537A>G in SMAD5 and g.319C>T in SMAD7, from Tibetan sheep, and they were genotyped. The expression patterns of SMAD4, SMAD5 and SMAD7 in the tissues of Tibetan sheep were determined. Furthermore, we also performed an association analysis of the SNPs with litter size. The results indicated that SMAD4, SMAD5 and SMAD7 were extensively expressed in the various tissues of Tibetan sheep. Sequence analysis showed that SMAD4 contained 1662 bp encoding 533 amino acids, SMAD5 contained 1667 bp encoding 465 amino acids and SMAD7 contained 1408 bp encoding 427 amino acids. Alignment analysis showed that the amino acid sequence of the three proteins shared high similarity with sheep, yak, cattle, dog, human, pig, chimpanzee, rhesus monkey and house mouse. Furthermore, association analysis showed that both g.51537A>G in SMAD5 and g.319C>T in SMAD7 were not significantly associated with litter size of Tibetan sheep. ABSTRACT: 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 SMAD4, SMAD5 and SMAD7 in Tibetan sheep and the effect of their polymorphism on litter size. In this study, we examined the expression of SMAD4, SMAD5 and SMAD7 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(TM) 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 SMAD4, SMAD5 and SMAD7 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 longissimus dorsi. However, the expression was unbalanced and upregulated in the spleen, lung, ovary and uterus and downregulated in the longissimus dorsi. The bioinformatics analysis showed that SMAD4, SMAD5 and SMAD7 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 SMAD4, SMAD5 and SMAD7 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 SMAD5 and SMAD7, 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 SMAD4, SMAD5 and SMAD7 in Tibetan sheep, but our results do not support associations between these genes and the litter size of Tibetan sheep.