佟, 彬Tong, Bin
153 , 2015-03-23 , 新潟大学
学位の種類: 博士（農学）. 報告番号: 甲第4053号. 学位記番号: 新大院博（農）甲第150号. 学位授与年月日: 平成27年3月23日
Marbling characterized by the amount and distribution of intramuscular fat in a cross section of musculus longissimus muscle is one of the economically important traits of beef cattle (JMGA, 1988). High levels of marbling improve the palatability and acceptability of beef by affecting the taste and tenderness of the meat (Busboom et al. 1993; Boylston et al. 1995; Matsuishi et al. 2001). Because of the importance of marbling on the economics of beef production, there is great interest in gaining a better understanding of the molecular architecture of marbling and generating new opportunities for more effective marker-assisted breeding. Although the statistical genetics, molecular genetics and genomic science have been utilized, the causal genes for the bovine marbling development are almost unidentified. To identify the genetic variation associated with marbling for improvement of beef quality through breeding program and to understand the biological network of marbling, two methods, quantitative trait locus (QTL) mapping and gene expression profiling for marbling have been performed (Georges & Andersson 1996; Georges 1997; Georges 1999; Andersson 2001; Andersson & Georges, 2003; Andersson & Georges 2004; Georges 2007). Yamada et al. applied the integrative approach combining mapping and expression profiling data to marbling study using Japanese Black beef cattle. They performed quantitative differential-display PCR (ddPCR) to examine differences in gene expression between high-marbled and low-marbled steers in musculus longissimus muscle (Sasaki et al. 2006b) and combined their expression profile data and the marbling QTL information. As a result, the endothelial differentiation sphingolipid G-protein-coupled receptor 1, titin, akirin 2, and ribosomal protein L27a genes showing marbling-associated expression changes in musculus longissimus muscle (Sasaki et al . 2006b) were found to be located within genomic region of marbling QTLs (Takasuga et al. 2007), which were mapped in a half-sib family of Japanese Black cattle (Yamada et al. 2006). Thus, these genes were regarded as positional functional candidates for the genes responsible for marbling. The SNPs detected in the promoter, 5’ untranslated or 3’ untranslated regions in these genes were associated with the predicted breeding value for beef marbling standard number by analyses using a population of Japanese Black beef cattle (Sasaki et al. 2009; Yamada et al. 2009a,b,c,d). These findings suggested that these SNPs may be useful for effective marker-assisted selection to increase the levels of marbling in Japanese Black beef cattle. However, these SNPs only explained around 15 percentages of genetic variance for marbling development, so we need to continue to identify more useful molecular markers for effective marker-assisted breeding. For this purpose, in this study, I undertook ddPCR to investigate gene-expression patterns associated with marbling in musculus longissimus muscle. And then, I searched functional annotations of candidate genes, utilized real-time PCR to confirm gene-expression pattern and performed QTL mapping database search as well as preliminary association study with closed marker to choose positional functional candidate genes. Further, I detected SNP in candidate gene and performed association analysis between SNP and marbling to select more useful molecular marker.
In 2nd Chapter, musculus longissimus muscle tissues were obtained from 2 Holstein steers as well as from 2 somatic nuclear-derived cloned steers from Itofuku, a Japanese Black sire with a very high predicted breeding value for marbling. The Holstein and cloned steers, respectively, had beef marbling standard numbers of 2 and 11 (potential range 1-12; JMGA 1988) at the time of slaughter at 29 months of age, representing low-marbled and high-marbled steers respectively. I utilized biopsies of musculus longissimus muscle from the steers at 8, 10, 12 and 14 months of age in order to encompass the 10 months at which marbling starts to appear. I performed quantitative ddPCR in order to detect differentially displayed bands between the low-marbled and high-marbled steer groups. A total of 2114 bands were identified on differentially displayed gels using 90 PCR primer pairs. Seventy-four bands showed putative differential expression patterns between low-marbled and high-marbled steer groups at 8, 10, 12 and 14 months of age. Of the 74 bands, 34, 28 and 12 bands, respectively, were significant (P < 0.05) for the group effect, the interaction effect between group and age, and both the group and the interaction effects.
In 3rd Chapter, 10 out of 74 differentially displayed bands were successfully excised from ddPCR gels, sequenced, and subjected to homology search. Homology search showed that 6 of the 10 unique sequences were known genes. Functional annotation search of the 6 genes were performed. As a result, the CDC10, TRDN, MFN2, MYBPC1, MYH1 and IRS1 genes, respectively, are known to be involved in cellular proliferation, to be involved in muscle contraction, to be mitochondrial protein that participates in mitochondrial fusion in mammalian cells and that is crucial to the maintenance and operation of the mitochondrial network and the mitochondrial metabolism in muscle cells, to be one isoform (in slow skeletal muscle) of myosin binding protein C that is one of the major myosin-binding proteins in vertebrate striated muscles, to encode an isoform of myosin heavy chain in type I (slow-oxidative) fiber of skeletal muscle, and to encode signaling adaptor that plays a major role in the metabolic and mitogenic actions of the insulin and insulin-like growth factors (IGF). And then, to validate the ddPCR results, real-time PCR was performed on the same steer total RNA samples as used in the quantitative ddPCR. The results of the real-time PCR for the expression patterns between the high-marbled Japanese Black steer group and low-marbled Holstein steer group of 6 genes were consistent with the quantitative ddPCR results. Based on the results of functional annotation search and expression patterns, the 6 genes could be considered as functional candidate gene for marbling development. In addition, I performed the QTL mapping database search and preliminary association study with closed marker to choose positional candidate gene for marbling development. All 6 genes were located in bovine chromosomal region of marbling QTL. As a result, the CDC10, TRDN, MFN2, MYBPC1, MYH1 and IRS1 genes could be considered as positional functional candidate gene for marbling development.
In 4th Chapter, I detected SNPs in the CDC10, TRDN, MFN2, MYBPC1, MYH1 and IRS1 genes between high-marbled steers and low-marbled steers using direct sequencing. There were 13 SNPs: CDC10-323, TRDN+412435, MFN2+1323, MFN2+1104, MFN2+1078, MFN2+ 810, MFN2+681, MFN2+1587, MYBPC1-4927, MYBPC1-4035, MYBPC1-3394, MYH1-2622 and IRS1-2910 have been detected. I established genotyping method based on PCR-restriction fragment length polymorphism (RFLP) for each SNP. Using this method, I performed preliminary association analysis using 34 Japanese Black unrelated sires (17 sires with extremely high predicted breeding value for marbling and 17 sires with extremely low one) selected from 100 unrelated sires. Only the MYBPC1-4927 SNP exhibited significant difference in the allele frequency distribution between 17 sires with extremely high breeding value and 17 sires with extremely low one. The frequency of the G allele at the MYBPC1-4927 SNP was higher in animals with extremely high breeding value than with extremely low one, and the A allele frequency in animals with the low one than with the high one. To investigate whether MYBPC1-4927 SNP was associated with marbling, I performed 2 experiments for the association study. I used 100 Japanese Black sires in experiment 1 and 745 paternal half-sib Japanese Black progeny steers produced from 2 sires (199 and 546 per sire) homozygous for A allele, with dams considered to be a random mating population, in experiment 2. Genotyping 100 sires for the SNP revealed 74 animals homozygous for the A allele, and 26 animals heterozygous for the A allele and the G allele in experiment 1. Statistically significant differences among the genotypes of the SNP were detected in the predicted breeding values for beef marbling standard number (P = 0.045). The predicted breeding values for beef marbling standard number were significantly higher in the AG heterozygotes than in the AA homozygotes at the MYBPC1-4927 SNP. To estimate better the effect of the SNP genotype on marbling, we used 745 progeny steers from 2 sires homozygous for the A allele at the MYBPC1-4927 SNP in experiment 2. These steers could be grouped according to the alleles that they received from their dams, allowing a linkage disequilibrium estimate of the effect of the SNP. The SNP genotype had statistically significant effect on the predicted breeding values for beef marbling standard number (P < 0.0001). Genotypic profiles of the predicted breeding values for beef marbling standard number were consistent with the result obtained by experiment 1. Based on the association of the MYBPC1-4927 with marbling, together with MYBPC1 expression difference between low-marbled steer group (with MYBPC1-4927 A allele) and high-marbled steer group (with MYBPC1-4927 A and G alleles), we hypothesized that the MYBPC1-4927 SNP might have an impact on MYBPC1 expression and also marbling by affecting MYBPC1 promoter activity. In conclusion, the information on the MYBPC1-4927 SNP obtained in this study, may be applied to effective marker-assisted selection to increase the levels of marbling in Japanese Black beef cattle.