Breed Discrimination Using DNA Markers Derived from AFLP in Japanese Beef Cattle

Sasazaki, S.,Imada, T.,Mutoh, H.,Yoshizawa, K.,Mannen, H. Asian Australasian Association of Animal Productio 2006 Animal Bioscience Vol.19 No.8

In the meat industry, correct breed information in food labeling is required to assure meat quality. Genetic markers provide corroborating evidence to identify breed. This paper describes the development of DNA markers to discriminate between Japanese Black and F1 (Japanese Black${\times}$Holstein) breeds. The amplified fragment length polymorphism method was employed to detect candidate markers absent in Japanese Black but present in Holstein. The 1,754 primer combinations yielded eleven markers that were converted into single nucleotide polymorphism markers for high-throughput genotyping. The allele frequencies in both breeds were investigated for discrimination ability using PCR-RFLP. The probability of identifying F1 was 0.9168 and probability of misjudgment was 0.0066 using four selected markers. The markers could be useful for discriminating between Japanese Black and F1 and would contribute to the prevention of falsified breed labeling of meat.

Mitochondrial DNA Variation and Genetic Relationships in Japanese and Korean Cattle

Sasazaki, S.,Odahara, S.,Hiura, C.,Mukai, F.,Mannen, H. Asian Australasian Association of Animal Productio 2006 Animal Bioscience Vol.19 No.10

The complete mtDNA D-loop regions of Japanese and Korean cattle were analyzed for their mtDNA variations and genetic relationships. Sequencing the 30 Higo substrain and 30 Tosa substrain of Japanese Brown, respectively 12 and 17 distinct Bos haplotypes were identified from 77 polymorphic nucleotide sites. In order to focus on the relationships among Japanese and Korean cattle, two types of phylogenetic tree were constructed using individual sequences; first, a neighbor-joining tree with all sequences and second, reduced median networks within each Japanese and Korean cattle group. The trees revealed that two major mtDNA haplotype groups, T3 and T4, were represented in Japanese and Korean cattle. The T4 haplogroup predominated in Japanese Black and Japanese Brown cattle (frequency of 43.3-66.7%), while the T3 haplogroup was predominant (83.3%) and T4 was represented only twice in the Korean cattle. The results suggested that the mitochondrial origins of Japanese Brown were Japanese ancient cattle as well as Japanese Black in despite of the considerable introgression of Korean and European cattle into Japanese Brown.

Genealogical Relationship between Pedigree and Microsatellite Information and Analysis of Genetic Structure of a Highly Inbred Japanese Black Cattle Strain

Sasazaki, S.,Honda, T.,Fukushima, M.,Oyama, K.,Mannen, H.,Mukai, F.,Tsuji, S. Asian Australasian Association of Animal Productio 2004 Animal Bioscience Vol.17 No.10

Japanese Black cattle of Hyogo prefecture (Tajima strain) are famous for its ability to produce high-quality meat and have been maintained as a closed system for more than 80 years. In order to assess the usefulness of microsatellite markers in closed cattle populations, and evaluate the genetic structure of the Tajima strain, we analyzed representative dams of the Tajima strain comprised of the substrains Nakadoi and Kinosaki. Genetic variability analyses indicated low genetic diversity in the Tajima strain. In addition, a recent genetic bottleneck, which could be accounted for by the high level of inbreeding, was detected in both substrains. In phylogenetic analyses, relationship coefficients and genetic distances between individuals were calculated using pedigree and microsatellite information. Two phylogenetic trees were constructed from microsatellite and pedigree information using the UPGMA method. Both trees illustrated that most individuals were distinguished clearly on the basis of the two substrains, although in the microsatellite tree some individuals appeared in clusters of different substrains. Comparing the two phylogenetic trees revealed good consistency between the microsatellite analysis tree and the pedigree information. The correlation coefficient between genetic distances derived from microsatellite and pedigree information was 0.686 with a high significance level (p<0.001). These results indicated that microsatellite information may provide data substantially equivalent to pedigree information even in unusually inbred herds of cattle, and suggested that microsatellite markers may be useful in revealing genetic structure without accurate or complete pedigree nformation. Japanese Black cattle of Hyogo prefecture (Tajima strain) are famous for its ability to produce high-quality meat and have been maintained as a closed system for more than 80 years. In order to assess the usefulness of microsatellite markers in closed cattle populations, and evaluate the genetic structure of the Tajima strain, we analyzed representative dams of the Tajima strain comprised of the substrains Nakadoi and Kinosaki. Genetic variability analyses indicated low genetic diversity in the Tajima strain. In addition, a recent genetic bottleneck, which could be accounted for by the high level of inbreeding, was detected in both substrains. In phylogenetic analyses, relationship coefficients and genetic distances between individuals were calculated using pedigree and microsatellite information. Two phylogenetic trees were constructed from microsatellite and pedigree information using the UPGMA method. Both trees illustrated that most individuals were distinguished clearly on the basis of the two substrains, although in the microsatellite tree some individuals appeared in clusters of different substrains. Comparing the two phylogenetic trees revealed good consistency between the microsatellite analysis tree and the pedigree information. The correlation coefficient between genetic distances derived from microsatellite and pedigree information was 0.686 with a high significance level (p<0.001). These results indicated that microsatellite information may provide data substantially equivalent to pedigree information even in unusually inbred herds of cattle, and suggested that microsatellite markers may be useful in revealing genetic structure without accurate or complete pedigree information.

Identification of the Gene Responsible for Chicken Muscular Dystrophy

Matsumoto, Hirokazu,Sasazaki, Shinji,Mannen, Hideyuki The Korean Society of Poultry Science 2011 韓國家禽學會誌 Vol.38 No.2

By a series of positional cloning, we successfully narrowed down the AM candidate region to approximately 1.2 Mbp on GGA2q including 7 functional genes. Subsequently, we identified WWP1 gene as the most likely AM candidate by sequence comparison. The amino acid sequence around the candidate mutation was highly conserved among tetrapods, suggesting that WWP1 is the causative gene of chicken muscular dystrophy. Transfection of mutated WWP1 gene into $C_2C_{12}$ myoblasts disrupted muscle differentiation process. The abnormal muscle differentiation is a characteristic of chicken muscular dystrophy, so we could demonstrate a part of phenotype of the disease. Furthermore, western blotting revealed that accumulation of caveolin-3 protein is limited in damaged muscle of muscular dystrophic chicken, suggesting caveolin-3 may be associated with the pathological change of the disease. We could conclude that WWP1 gene is the responsible one for chicken muscular dystrophy from these results, but the mechanism leading the onset should be clarified in the future. The information will contribute to the study of chicken muscular dystrophy and the corresponding human dystrophies.

Proteomic Comparison between Japanese Black and Holstein Cattle by Two-dimensional Gel Electrophoresis and Identification of Proteins

Ohsaki, H.,Okada, M.,Sasazaki, S.,Hinenoya, T.,Sawa, T.,Iwanaga, S.,Tsuruta, H.,Mukai, F.,Mannen, H. Asian Australasian Association of Animal Productio 2007 Animal Bioscience Vol.20 No.5

Differences of meat qualities between Japanese Black and Holstein have been known in Japan, however, the causative proteins and/or the genetic background have been unclear. The aim of this study was to identify candidate proteins causing differences of the meat qualities between the two breeds. Using technique of two-dimensional gel electrophoresis, protein profiling was compared from samples of the longissimus dorsi muscle and subcutaneous adipose tissue. Five protein spots were observed with different expression levels between breeds. By using LC-MS/MS analysis and Mascot program, three of them were identified as ankyrin repeat protein 2, phosphoylated myosin light chain 2 and mimecan protein. Subsequently, we compared the DNA coding sequences of three proteins between breeds to find any nucleotide substitution. However, there was no notable mutation which could affect pI or molecular mass of the proteins. The identified proteins may be responsible for different characteristics of the meat qualities between Japanese Black and Holstein cattle.

Identification of the Gene Responsible for Chicken Muscular Dystrophy

Hirokazu Matsumoto,Shinji Sasazaki,Hideyuki Mannen 韓國家禽學會 2011 韓國家禽學會誌 Vol.38 No.2

By a series of positional cloning, we successfully narrowed down the AM candidate region to approximately 1.2 Mbp on GGA2q including 7 functional genes. Subsequently, we identified WWP1 gene as the most likely AM candidate by sequence comparison. The amino acid sequence around the candidate mutation was highly conserved among tetrapods, suggesting that WWP1 is the causative gene of chicken muscular dystrophy. Transfection of mutated WWP1 gene into C₂C₁₂ myoblasts disrupted muscle differentiation process. The abnormal muscle differentiation is a characteristic of chicken muscular dystrophy, so we could demonstrate a part of phenotype of the disease. Furthermore, western blotting revealed that accumulation of caveolin-3 protein is limited in damaged muscle of muscular dystrophic chicken, suggesting caveolin-3 may be associated with the pathological change of the disease. We could conclude that WWP1 gene is the responsible one for chicken muscular dystrophy from these results, but the mechanism leading the onset should be clarified in the future. The information will contribute to the study of chicken muscular dystrophy and the corresponding human dystrophies.

Mitochondrial DNA Diversity of Korean Native Goats

Odahara, S.,Chung, H.J.,Choi, S.H.,Yu, S.L.,Sasazaki, S.,Mannen, H.,Park, C.H.,Lee, J.H. 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10

Korean native goats have lived on the Korean peninsula for more than 2,000 years and are regarded as a valuable genetic resource for the world. As an initial step to investigate the genetic structures of this breed, phylogenetic analysis and calculation of genetic diversities have been performed using mitochondrial DNA (mtDNA) sequence variations. A total of 19 Korean native goats were grouped into six haplotypes and the large majority of haplotypes were present in 13 animals. All mtDNA of these Korean goats belonged to the mitochondrial (mt) lineage A and revealed remarkably small genetic distances within the population when compared with other Asian goat populations, indicating less genetic variation in the Korean native goats. These results indicate high-inbred status of the Korean native goats and will influence breeding and conservation strategies adopted for this breed.

Mitochondrial DNA Diversity of Korean Native Goats

Odahara, S.,Chung, H.J.,Choi, S.H.,Yu, S.L.,Sasazaki, S.,Mannen, H.,Park, C.S.,Lee, J.H. Asian Australasian Association of Animal Productio 2006 Animal Bioscience Vol.19 No.4

Korean native goats have lived on the Korean peninsula for more than 2,000 years and are regarded as a valuable genetic resource for the world. As an initial step to investigate the genetic structures of this breed, phylogenetic analysis and calculation of genetic diversities have been performed using mitochondrial DNA (mtDNA) sequence variations. A total of 19 Korean native goats were grouped into six haplotypes and the large majority of haplotypes were present in 13 animals. All mtDNA of these Korean goats belonged to the mitochondrial (mt) lineage A and revealed remarkably small genetic distances within the population when compared with other Asian goat populations, indicating less genetic variation in the Korean native goats. These results indicate high-inbred status of the Korean native goats and will influence breeding and conservation strategies adopted for this breed.

DNA marker-based breed identification in cattle

Kwon, Y.S.,Yoon, D.H.,Lee, K.Y.,Jung, W.Y.,Sasazaki, S.,Mannen, H.,Jeon, J.T.,Lee, J.H. 충남대학교 형질전환복제돼지연구센터 2007 논문집 Vol. No.10

Discrimination of Korean Cattle (Hanwoo) Using DNA Markers Derived from SNPs in Bovine Mitochondrial and SRY Genes

Yoon, D.,Kwon, Y.S.,Lee, K.Y.,Jung, W.Y.,Sasazaki, S.,Mannen, H.,Jeon, J.T.,Lee, J.H. Asian Australasian Association of Animal Productio 2008 Animal Bioscience Vol.21 No.1

In order to distinguish Korean cattle (Hanwoo) beef from the imported beef from Australia in Korean markets, DNA markers based on PCR-RFLP from mitochondrial genes and SRY gene were applied. A total of 2,826 beef samples comprising 1,495 Hanwoo and 1,331 foreign cattle breeds were obtained in Korea. An 801 bp fragment of the SRY gene on the bovine Y chromosome, a 343 bp fragment of ND4 gene and a 528 bp fragment of ND5 gene in the bovine mtDNA were amplified by PCR and digested with three restriction enzymes, MseI, HpyCH4III and Tsp509I, respectively. The results showed that Bos taurus (T) type was the majority in Hanwoo by combining three markers (99.5%). However, 78.2% of Bos indicus (I) type was observed in the imported beef samples. These results indicated that three markers used in this study will be used as valuable markers for discriminating imported beef against Hanwoo.