DNA methylation analysis of exon-1 of the ovine HOXC-8 gene in Mongolian sheep using bisulphite sequencing

Abstract

The multi-vertebrae phenotype of the Mongolian sheep is not explained by Mendel's law. To test the hypothesis that the multi-vertebrae phenotype is related to HOXC-8 mutation or genomic imprinting, the sequences of exon-1 and exon-2 of the HOXC-8 gene of normal and multi-vertebrae Mongolian sheep were analysed. Bisulphite sequencing Polymerase Chain Reaction (PCR) was employed to compare the methylation status of cytosine-guanine (CpG) sites in HOXC-8 exon-1 region from normal and multi-vertebrae Mongolian sheep. No mutations were observed in HOXC-8 exon-1 or exon-2 of normal and multi-vertebrae Mongolian sheep. However, the level of DNA methylation in exon-1 of the HOXC-8 gene was much lower in the 13-vertebrae sheep than in the 14-vertebrae sheep. In the CpG island (293 bp), there were six, three and six methylated CpGs in the normal sheep samples and 23, 20 and 21 in the multi-vertebrae sheep samples, respectively. The ratio of methylated to non-methylated CpG dinucleotides in normal and multi-vertebrae sheep were 18.50±0.064 and 79.03±0.056% (P<0.01, by a t-test), respectively.

Keywords:

• bisulphite sequencing PCR
• DNA methylation
• multi-vertebrae phenotype

Acknowledgements

This work was supported by the 11th Five-Year Plan National Science and Technology Support Program of China (No. 2006BDA13B08) and National Natural Science Foundation of China (No. 30960245).