Molecular characterization and expression of TGFßRI and TGFßRII and its association with litter size in Tibetan sheep.

BACKGROUNDS: Transforming growth factor-ß (TGF-ß) type I receptor (TGFßRI) and type II receptor (TGFßRII) are the members of the TGFß superfamily, which are potent regulators of cell proliferation and differentiation in many organ systems, and they play key roles in multiple aspects of follicle development. OBJECTIVES: We aimed to explore the characterization, expression analysis of TGFßRI and TGFßRII genes, and the association with litter size in Tibetan sheep. METHODS: In this study, we cloned the complete coding sequences of TGFßRI and TGFßRII genes in Tibetan sheep and analyzed their genomic structures. RESULTS: The results showed that percentages of sequences homology of the two proteins in Tibetan sheep were the most similar to Ovis aries (100%), followed by Bos mutus (99%). The RT-qPCR showed that two genes were expressed widely in the different tissues of Tibetan sheep. The TGFßRI expression was the highest in the lung (p < 0.05), followed by the spleen and ovary (p < 0.05). The TGFßRII expression was significantly higher in uterus than that in lung and ovary (p < 0.05). In addition, the χ2 test indicated that all ewes in the population were in Hardy-Weinberg equilibrium, and the population was in medium or low polymorphic information content status. We also found four Single Nucleotide Polymorphism (SNPs), g.9414A > G, g.28881A > G, g.28809T > C, g.10429G > A in sheep TGFßRI gene and g.63940C > T, g.63976C > T, g.64538C > T, g.64504T > A in TGFßRII gene. Three genotypes, except for g.64504T > A, and three haplotypes were identified in each gene. linkage disequilibrium analysis indicated that there was strong linkage disequilibrium in each gene. The association analysis showed that the four SNPs of TGFßRI were associated with litter size (p < 0.05), and g.63940C > T of TGFßRII was confirmed to be associated with litter size (p < 0.05). CONCLUSIONS: Based on these preliminary results, we can assume that TGFß receptors (TGFßRI and TGFßRII) may play an important role in sheep reproduction.

Expression and Polymorphisms of SMAD1, SMAD2 and SMAD3 Genes and Their Association with Litter Size in Tibetan Sheep (Ovis aries).

SMAD1, SMAD2, and SMAD3 are important transcription factors downstream of the TGF-ß/SMAD signaling pathway that mediates several physiological processes. In the current study, we used cloning sequencing, RT-qPCR, bioinformatics methods and iMLDR technology to clone the coding region of Tibetan sheep genes, analyze the protein structure and detect the tissue expression characteristics of Tibetan sheep genes, and detect the polymorphisms of 433 Tibetan sheep and analyze their correlation with litter size. The results showed that the ORFs of the SMAD1, SMAD2 and SMAD3 genes were 1398 bp, 1404 bp and 1278 bp, respectively, and encoded 465, 467 and 425 amino acids, respectively. The SMAD1, SMAD2, and SMAD3 proteins were all unstable hydrophilic mixed proteins. SMAD1, SMAD2 and SMAD3 were widely expressed in Tibetan sheep tissues, and all were highly expressed in the uterus, spleen, ovary and lung tissues. Litter sizes of the genotype CC in the SMAD1 gene g.10729C>T locus were significantly higher than that of CT (p < 0.05). In the SMAD3 gene g.21447C>T locus, the genotype TT individuals showed a higher litter size than the CC and CT genotype individuals (p < 0.05). These results preliminarily demonstrated that SMAD1, SMAD2 and SMAD3 were the major candidate genes that affected litter size traits in Tibetan sheep and could be used as a molecular genetic marker for early auxiliary selection for improving reproductive traits during sheep breeding.

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

SMAD4, SMAD5 and SMAD7 belonging to the transforming growth factor ß (TGF-ß) 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-LDRTM 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.

Meadow degradation increases spatial turnover rates of the fungal community through both niche selection and dispersal limitation.

The alpine meadow ecosystem, as the main ecosystem of the Qinghai-Tibet Plateau, has been heavily degraded over the past several decades due to overgrazing and climate change. Although soil microorganisms play key roles in the stability and succession of grassland ecosystems, their response to grassland degradation has not been investigated at spatial scale. Here, we systematically analyzed the spatial turnover rates of soil prokaryotic and fungal communities in degraded and undegraded meadows through distance-decay relationship (DDR) and species area relationship (SAR), as well as the community assembly mechanisms behind them. Although the composition and structure of both fungal and prokaryotic communities showed significant changes between undegraded and degraded meadows, steeper spatial turnover rates were only observed in fungi (Degraded Alpine Meadow ß = 0.0142, Undegraded Alpine Meadow ß = 0.0077, P < 0.05). Mantel tests indicated that edaphic variables and vegetation factors showed significant correlations to the ß diversity of fungal community only in degraded meadow, suggesting soil and vegetation heterogeneity both contributed to the variation of fungal community in that system. Correspondingly, a novel phylogenetic null model analysis demonstrated that environmental selection was enhanced in the fungal community assembly process during meadow degradation. Interestingly, dispersal limitation was also enhanced for the fungal community in the degraded meadow, and its relative contribution to other assembly process (i.e. selection and drift) showed a significant linear increase with spatial distance, suggesting that dispersal limitation played a greater role as distance increased. Our findings indicated the spatial scaling of the fungal community is altered during meadow degradation by both niche selection and dispersal limitation. This study provides a new perspective for the assessment of soil microbial responses to vegetation changes in alpine areas.

Genetic variation and relationships in the mitochondrial DNA D-loop region of Qinghai indigenous and commercial pig breeds.

BACKGROUND: Bamei pigs are special germplasm resources in Qinghai. In the specific environment of the Qinghai plateau, local breeds, through long-term natural and artificial selection, have acquired a relatively stable population structure and genetic diversity. This study revealed Bamei pigs' genetic diversity at the molecular level and the degree of introgression of commercial breeds into Bamei pigs. METHODS: The mitochondrial DNA D-loop region was amplified and sequenced using the ABI 3130 DNA sequencer. Sequences were collected, edited and aligned using the MEGA 5.0, DNASTAR and ClustalX 1.81 software. Haplotypes of all sequences and genetic diversity were analyzed by DnaSP 5.0 software. RESULTS AND CONCLUSIONS: We identified a total of 73 polymorphic sites in our 489 novel sequences, including 1 single variable site and 72 parsimony informative sites.Genetic diversity was analyzed in this study. The results showed that haplotype diversity, nucleotide diversity and the average number of nucleotide differences of Bamei pigs were lower than those of commercial pigs. Synthetic evaluation of genetic diversity through principal component analysis indicated that Bamei pigs also showed low genetic diversity. In summary, the results of sequence analysis showed that Bamei pigs were low in genetic diversity, and the introgression of commercial pigs into Bamei pigs was serious.