| Research Abstract |
It is unique that our expression profiling-based strategy strategy utilized somatic nuclear-derived clones of Itofuku, a superior sire with the very highly predicted breeding value for marbling as a high-marbled group. Biopsy samples of the longissimus-dorsi muscle were taken from two groups of the high-marbled and the low-marbled Holstein steers at 8,10,12 and 14 months of age. Two types of differential display-PCRs were applied to explore genes of which expression pattern is different and coincident with the process of intramuscular fat accumulation between the two steer groups. A total of 2,114 bands were detected on differentially displayed gels. Of these bands, 74 bands showed the expression pattern related to intramuscular fat accumulation by utilizing the two method. Sequence analysis of the 74 differentially displayed bands revealed 77 genes, including 35 annotated and 42 novel genes.
Subsequently, we identified candidates for beef marbling genes among the 77 genes by performing
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retrieval of known gene function in silico and analysis of expression pattern with real-time quantitative PCRs. Of the 35 annotated genes, 12 (BTG2,CDC10,MAF,WBP2,SORBS1,PDHB,VAPA,SLC30A6,TTN,NEB,TRDN,EDG1) were identified as the genes with possible functions related to intramuscular fat accumulation. Further, the 5 (BTG2,WBP2,VAPA,TTN, and EDG1) of the 12 genes exhibited expression patterns coincident with the process of intramuscular fat accumulation, and thus were considered as candidates for beef marbling genes. In addition, one of these five genes, EDG1, was localized using radiation hybrid mapping within the chromosomal region of a marbling QTL detected in Japanese Black breed. Taken together, we supposed that the EDG1 was regarded as a candidate gene in terms of its position as well as its function and expression pattern.
Finally, we clarified genomic structure of the candidate genes, detected the SNPs in the genes, and studied the association of its SNPs with beef marbling trait. Genomic structures of the BTG2, WBP2, VAPA, and EDG1 genes were clarified by cloning and sequencing. Two (-71 bp and -1,702 bp in promoter region), three (-1,723 bp in promoter region, +524 bp in coding region, and +1,192 bp in 3'-untranslated region), and two (-87 bp in promoter region and +3,258 bp in 3'-untranslated region) SNPs were found in the WBP2, VAPA, and EDG1, respectively, between high-marbled Japanese Black cattle and low-marbled Holstein cattle, which were used for expression profiling. Any SNPs were not detected in the BTG2. The SNPs in the WBP2 and VAPA showed no association with marbling trait. Interestingly, a haplotype comprising G allele at -87 bp and A allele at +3,258 bp in the EDG1 was associated with high-marbled phenotype in Japanese Black breed, while another comprising -87 bp A allele and +3,258 G allele with low-marbled phenotype. Therefore, it is reasonable to conclude that the EDG1 gene is responsible for controlling beef marbling in Japanese Black cattle breed. Less
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