Identification of the genes responsible for beef marbling using somatic nuclear cloning technology

独一无二的是，我们的基于表达谱的策略策略利用了伊藤福库的体细胞核克隆，这是一个具有非常高的大理石花纹育种价值的优良父系，作为一个高大理石花纹群体。取8、10、12、14个月龄的高大理石花纹和低大理石花纹荷斯坦牛两组背最长肌的活组织标本。应用两种差异显示-PCR技术，筛选出两种不同的、与肌内脂肪堆积过程相吻合的基因表达模式。在差异显示的凝胶上共检测到2,114条条带。在这些条带中，有74条条带显示了与肌内脂肪积累相关的表达模式。对74条差异显示的条带进行序列分析，共发现77条基因，其中35条为注释基因，42条为新基因，通过…从77条基因中筛选出牛肉大理石纹基因的候选基因在计算机中更多地检索已知基因的功能，并用实时定量PCRS分析表达模式。在35个注释基因中，12个(BTG2、CDC10、MAF、WBP2、SORBS1、PDHB、VAPA、SLC30A6、TTN、NEB、TRDN、EDG1)被鉴定为可能与肌内脂肪积累有关的基因。此外，12个基因中的5个(BTG2、WBP2、VAPA、TTN和EDG1)表现出与肌肉内脂肪积累过程一致的表达模式，因此被认为是牛肉大理石纹基因的候选基因。此外，这五个基因中的一个，EDG1，通过辐射杂交定位在日本黑种大理石纹QTL的染色体区域内。综上所述，我们认为EDG1基因的定位、功能和表达模式可能是一个候选基因。最后，我们明确了候选基因的基因组结构，检测了基因中的SNPs，并研究了其SNPs与牛肉大理石纹性状的关系。通过克隆和测序明确了BTG2、WBP2、VAPA和EDG1基因的基因组结构。在高大理石纹日本黑牛和低大理石纹荷斯坦牛的WBP2、VAPA和EDG1基因序列中，分别检测到2个(-71和-1,702个碱基)、3个(启动子-1,723个碱基、编码区+524个和3‘-非翻译区+1,192个碱基)和2个(启动子-87个和3’-非翻译区+3,258个碱基)SNPs。在BTG2中没有检测到任何SNP。WBP2和VAPA中的SNPs与大理石花纹性状无关。有趣的是，EDG1中包含G等位基因和+3,258bp的G等位基因的单倍型与日本黑猪大理石纹高的表型相关，而另一个单倍型的-87bP的A等位基因和+3,258的G等位基因与低大理石纹的表型相关。因此，EDG1基因对日本黑牛大理石纹的控制作用是合理的。较少

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 … More 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