Japanese Native Breeds Research Articles

A Nonsynonymous Substitution of Lhx3 Leads to Changes in Body Size in Dogs and Mice.

Lhx3 is a LIM-homeodomain transcription factor that affects body size in mammals by regulating the secretion of pituitary hormones. Akita, Shiba Inu, and Mame Shiba Inu dogs are Japanese native dog breeds that have different body sizes. To determine whether Lhx3 plays a role in the differing body sizes of these three dog breeds, we sequenced the Lhx3 gene in the three breeds, which led to the identification of an SNP in codon 280 (S280N) associated with body size. The allele frequency at this SNP differed significantly between the large Akita and the two kinds of smaller Shiba dogs. To validate the function of this SNP on body size, we introduced this change into the Lhx3 gene of mice. Homozygous mutant mice (S279N+/+) were found to have significantly increased body lengths and weights compared to heterozygous mutant (S279N+/-) and wild-type (S279N-/-) mice several weeks after weaning. These results demonstrate that a nonsynonymous substitution in Lhx3 plays an important role in regulating body size in mammals.

Genetic characterization of Japanese native horse breeds by genotyping variants that are associated with phenotypic traits.

Concerns have been raised about the loss of genetic diversity in Japanese native horses because of their declining populations. In this study, we investigated the genetic variation of four genes, myostatin (MSTN), ligand-dependent nuclear receptor corepressor like (LCORL), doublesex and mab-3 related transcription factor 3 (DMRT3), and 5-hydroxytryptamine receptor 1A (HTR1A), which are associated with horse phenotypic traits, in six Japanese horse breeds (Hokkaido, Kiso, Noma, Misaki, Tokara, and Yonaguni). MSTN, LCORL, DMRT3, and HTR1A showed polymorphisms in the Kiso; Hokkaido and Noma; Hokkaido; and Kiso, Tokara, and Yonaguni breeds, respectively. The Misaki did not show polymorphisms in any of the genes. This study may serve as a basis for developing future breeding strategies focusing on traits in Japanese native horses.

Genetic diversity analysis and parentage verification of Taishu horses using 31 microsatellites.

The Taishu horse in Tsushima is one of eight Japanese native breeds. The breed is on the verge of extinction due to a rapid decrease in numbers since the 1960s owing to motorization in Japan. In this study, we aimed to confirm the pedigree information of 52 horses by genotyping 31 microsatellites in order to avoid inbreeding. Parentage verification failed to identify genetic contradictions among trios (sires, dams, and foals) registered with the Japan Equine Affairs Association (JEAA). Pedigree information registered at the JEAA was obtained and adequately understood. Additionally, the genetic diversity of the Taishu horses was evaluated and compared with those of other Japanese native breeds. The average values for the number of alleles, observed heterozygosity, expected heterozygosity, and inbreeding coefficient were 4.7, 0.643, 0.632, and -0.02, respectively. Using the Structure software, the 52 horses were classified into three subgroups based on the individuals with more than 50% of specific genetic components. The phylogenetic trees created based on neighbor-joining classification tended to be consistent among the stallions. The effective population size was 27.5 and lower than that required for maintaining 90% genetic variation in the source population over a period of 100 years (47.5). Compared with the other Japanese breeds, the Taishu horse population included in the current study exhibited moderate genetic diversity. Our study will contribute to reconsideration of the breeding strategy of Taishu horses.

A conserved haplotype in Wagyu cattle contains RAB4A whose encoded protein regulates glucose trafficking in muscle and fat cells

The Wagyu breed of taurine cattle possess favourable genetics for intramuscular fat (IMF) but genomic loci associated with the trait remain under characterised. Here, we report the identification of a previously unidentified genomic region possessing a particular haplotype structure in Wagyu. Through deployment of a genome-wide haplotype detection analysis that captures regions conserved in a target population but not other populations we screened 100 individual Wagyu and contrasted them with 100 individuals from two independent comparison breeds, Charolais and Angus, using high-density SNPs. An extreme level of Wagyu conservation was assigned to a single genomic window (spanning genomic coordinates BTA28:41088-300265bp). In fact, a five-SNP region spanning 27096bp is almost perfectly conserved among the 100 Wagyu individuals assayed and partially overlaps RAB4A. Focussing in, two consecutive SNPs (genomic coordinates 236949 and 239950) are apparently fixed within the Wagyu (BB and AA respectively), but at mixed frequencies in the other two breeds. These SNPs are located in the two introns straddling exon 7. In a separate analysis using the 1000 Bulls database, we found that, coincident with exon 7 of RAB4A first allele frequencies were highest in the high IMF Japanese Native (Wagyu) breeds (0.78) and lowest in the low IMF indicine breeds (Nelore and Brahman), with intermediate marbling breeds (Angus and Charolais) assigned intermediate rankings (0.42). RAB4A is known to encode a protein that regulates intracellular trafficking of the insulin-regulated glucose transporter GLUT4. RAB4A can be considered an attractive new positional candidate for IMF development.

The use of behavioral tests of fearfulness in chicks to distinguish between the Japanese native chicken breeds, Tosa-Kukin and Yakido.

The aim of this study was to investigate the differences in fearfulness between two Japanese native chicken breeds, Tosa-Kukin (TOK) and Yakido (YKD). In a tonic immobility test, TOK breed chicks showed longer duration and lower induction number compared with YKD. The number of peeps in YKD in an isolation test was lower than that in TOK, whereas there were more bouts of peeping in YKD than in TOK. In a manual restraint test, YKD showed more active responses and initiated peeps and struggling earlier than TOK. The three behavioral tests all indicated that YKD are less fearful than TOK chickens. A latent structures discriminant (OPLS-DA) analysis was used to identify behavioral parameters that contributed to the differences between the breeds. The major parameters were duration and number of inductions in the tonic immobility test and number of struggle in the manual restraint test. These results suggest that three behavioral tests can be used together to evaluate fearfulness of Japanese native breeds of chickens.

Genetic diversity and relationships among native Japanese horse breeds, the Japanese Thoroughbred and horses outside of Japan using genome-wide SNP data.

Eight horse breeds-Hokkaido, Kiso, Misaki, Noma, Taishu, Tokara, Miyako and Yonaguni-are native to Japan. Although Japanese native breeds are believed to have originated from ancient Mongolian horses imported from the Korean Peninsula, the phylogenetic relationships among these breeds are not well elucidated. In the present study, we compared genetic diversity among 32 international horse breeds previously evaluated by the Equine Genetic Diversity Consortium, the eight Japanese native breeds and Japanese Thoroughbreds using genome-wide SNP genotype data. The proportion of polymorphic loci and expected heterozygosity showed that the native Japanese breeds, with the exception of the Hokkaido, have relatively low diversity compared to the other breeds sampled. Phylogenetic and cluster analyses demonstrated relationships among the breeds that largely reflect their geographic distribution in Japan. Based on these data, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The Japanese Thoroughbreds were distinct from the native breeds, and although they maintain similar overall diversity as Thoroughbreds from outside Japan, they also show evidence of uniqueness relative to the other Thoroughbred samples. This is the first study to place the eight native Japanese breeds and Japanese Thoroughbred in context with an international sample of diverse breeds.

Genetic analysis of Taishu horses on and off Tsushima Island: Implications for conservation.

ABSTRACTTaishu horses are a native Japanese breed, of which only 41 individuals remained on Tsushima Island in 2018. Their genetic diversity is considered lower than that of other Japanese native horse breeds; thus, it needs to be investigated for sustainable conservation of this breed. Historical records revealed that several Taishu individuals were released areas off-Tsushima Island in mid-1980s. At present, Taishu horses living outside of Tsushima Island, hereafter referred to as Non-Tsushima Taishus (NTTs), are tagged. However, the genetic structure of the NTT individuals remains unclear, and such individuals are not included in the current mating plans for Taishu horses. Herein, we examined the genetic structure of 18 NTT individuals by comparing their genomic (SNP) information with that of individuals on Tsushima Island (TT), four other native Japanese breeds, and one Anglo-Arabian breed by using ddRAD-seq. We found that all individuals related to the Taishu can be grouped in one cluster, which was separated from other horse breeds. Patterns of specific and shared SNPs in NTT individuals closely resembled those of TT individuals, suggesting very minor genetic differences. Meanwhile, the heterozygosity of NTT individuals was slightly higher than that of TT individuals, and many NTT individuals were of fertile age, suggesting that the pedigree of NTT individuals would be useful in breed conservation plans for Taishu horses. Based on their genomic information, we also reconstructed the pedigree structures of four NTT individuals with no family information. The inclusion of NTT individuals in future mating plans on Tsushima Island may be an effective and feasible method for conserving the Taishu horse breed in Japan.

Genome-wide association mapping and examination of possible maternal effect for the pace trait of horses.

Previously, a single nucleotide polymorphism (SNP) related to gait type was identified at position 22999655 of chromosome 23 in the coding region of DMRT3 (DMRT3:Ser301Ter) by showing that a cytosine (C) to adenine (A) mutation of this SNP induced pace in the Icelandic horse. We investigated the effect of DMRT3:Ser301Ter on the gait of Hokkaido Native Horses, a Japanese native breed, and examined genetic factors other than DMRT3 by exploring genome-wide SNPs related to gait determination. All animals exhibiting pace were AA for DMRT3:Ser301Ter, confirming the association of DMRT3:Ser301Ter with gait determination; however, 14.3% of the animals exhibiting trot also had AA for DMRT3:Ser301Ter, suggesting the presence of another factor(s) cooperatively working with DMRT3:Ser301Ter for gait determination. SNPs on chromosomes 13 and 23 were detected by genome-wide association analysis (false discovery rate <0.05), although SNPs on chromosome 23 were all located in the vicinity of DMRT3:Ser301Ter, confirming the association with DMRT3. A genome-wide association study targeting only animals with AA for DMRT3:Ser301Ter to examine genetic factors cooperatively working with DMRT3:Ser301Ter for gait determination suggested associations of 23 SNPs on six chromosomes. In a series of analyses of the effect of a maternal factor (dam's gait) on gait determination, the effect was suggested in comparison of the frequencies of exhibiting pace in gait checks in only two animal groups having dams with different DMRT3:Ser301Ter genotypes (P<0.05), suggesting that the gait of the dam does not have a major effect on whether progeny homozygous for the DMRT3:Ser301Ter mutation will preferentially pace or trot.

Investigation of genetic diversity and inbreeding in a Japanese native horse breed for suggestions on its conservation.

Because native breeds can serve as genetic resources for adapting to environment changes, their conservation is important for future agroecosystems. Using pedigree analysis, we investigated genetic diversity and inbreeding in Japanese Hokkaido native horses, which have adapted to a cold climate and roughage diet. Genetic diversity was measured as the number of founders and the effective number of founders, ancestors and genomes. All metrics imply a decrease in genetic diversity. A comparison of these metrics suggested that pedigree bottlenecks contributed more than did random gene losses to the reduction of genetic diversity. Estimates of marginal contributions of ancestors suggest that the bottlenecks arose mainly because related stallions had been used for breeding. A tendency for an increase in inbreeding coefficients was observed. F-statistics revealed that a small effective population size majorly contributed to this increase, although non-random mating in particular regions also contributed. Because the bottlenecks are thought to have reduced the effective population size, our results imply that mitigation of bottlenecks is important for conservation. To this end, breeding should involve genetically diverse stallions. In addition, to prevent non-random mating observed in particular regions, efforts should be made to plan mating with consideration of kinships.

Is egg flavour changeable by chicken breeding? Association of chicken fatty acid desaturase 1 gene single-nucleotide polymorphisms with egg fatty acid profiles and flavour in a Japanese hybrid chicken

AbstractIn Japan, hybrid crosses between Japanese native and exotic breeds, known as “Jidori” chickens, are sold at high prices to a limited market. Most Japanese recognize that eggs produced from Jidori chickens have a rich flavour, named kokumi, whereas the reason that Jidori eggs are kokumi-rich has not yet been elucidated. The main objective in this study was to elucidate why Jidori eggs have a rich flavour. We investigated whether polymorphisms in fatty acid desaturase 1 and 2 (FADS1 and FADS2) affected the fatty acid profiles of Suruga-shamo eggs, a chicken breed in Japan. A single-nucleotide polymorphism (SNP) in FADS1 was significantly associated with arachidonic acid (ARA) content. The n − 6/n − 3 polyunsaturated fatty acid ratio of the yolk showed that the SNP allele that exhibited high ARA had a low n − 6/n − 3 ratio. We found that adding trace amounts of ARA, that correspond to the difference of the SNP genotypes, enhanced egg flavour intensity and continuity. We concluded that the SNP of FADS...

Copy number variations in the amylase gene (AMY2B) in Japanese native dog breeds.

A recent study suggested that increased copy numbers of the AMY2B gene might be a crucial genetic change that occurred during the domestication of dogs. To investigate AMY2B expansion in ancient breeds, which are highly divergent from modern breeds of presumed European origins, we analysed copy numbers in native Japanese dog breeds. Copy numbers in the Akita and Shiba, two ancient breeds in Japan, were higher than those in wolves. However, compared to a group of various modern breeds, Akitas had fewer copy numbers, whereas Shibas exhibited the same level of expansion as modern breeds. Interestingly, average AMY2B copy numbers in the Jomon-Shiba, a unique line of the Shiba that has been bred to maintain their appearance resembling ancestors of native Japanese dogs and that originated in the same region as the Akita, were lower than those in the Shiba. These differences may have arisen from the earlier introduction of rice farming to the region in which the Shiba originated compared to the region in which the Akita and the Jomon-Shiba originated. Thus, our data provide insights into the relationship between the introduction of agriculture and AMY2B expansion in dogs.

Genetic diversity of maternal lineage in the endangered Kiso horse based on polymorphism of the mitochondrial DNA D-loop region.

ABSTRACTTo determine genetic characteristics of the maternal lineage of the Kiso horse based on polymorphisms of the mitochondrial DNA D-loop region, we collected blood samples from 136 Kiso horses, 91% of the entire population, and sequenced 411 bp from 15,437 to 15,847 in the region. First of all, we estimated the demographic history; by searching homology between the obtained and known sequences using Basic Local Alignment Search Tool, by mismatch analysis to evaluate the mutation processes using Arlequin, and by building a phylogenetic tree showing the relationship of the mtDNA haplotypes for 24 horse breeds around the world using Molecular Evolutionary Genetics Analysis softwear. The results suggested that various horses that came to Japan stayed at Kiso region and became ancestors of Kiso horse and also genetically supported the theory that the Kiso horse was historically improved by other Japanese native horse breeds. Next, we analyzed the diversity of current maternal lineage by classifying the resulting sequences, and by calculating the haplotype diversity and nucleotide diversity using Arlequin. Then, we visualized the relationship among haplotypes by a median-joining network using NETWORK 4.6.0.0. The results suggested the diversity of maternal lineage in the Kiso horse was reasonably maintained. Lastly, we predicted future change of the diversity of maternal lineage in Kiso horse by assessing the regional distribution of the acquired haplotypes. The distribution suggested that diversity of maternal lineage would possibly be reducing.

Genetic relationships between Japanese native and commercial breeds using 70 chicken autosomal SNP genotypes by the DigiTag2 assay

Recently, single nucleotide polymorphisms (SNPs) have been used to identify genes or genomic regions responsible for economic traits, including genetic diseases in domestic animals, and to examine genetic diversity of populations. In this study, we genotyped 70 chicken autosomal SNPs using DigiTag2 assay to understand the genetic structure of the Japanese native chicken breeds Satsumadori and Ingie, and the relationship of these breeds with other established breeds, Rhode Island Red (RIR), commercial broiler and layer. Five breeds, each consisting of approximately 20 chickens, were subjected to the assay, revealing the following: Average expected heterozygosities of broiler, Satsumadori, RIR, layer and Ingie were 0.265, 0.254, 0.244, 0.179 and 0.176, respectively. Phylogenetic analysis using the concatenated 70 autosomal SNP genotypes distinguished all chickens and formed clusters of chickens belonging to the respective breeds. In addition, the 2-D scatter plot of the first two principal components was consistent with the phylogenic tree. Taken together with the pairwise F(st) distances, broiler and RIR were closely positioned near each other, while Ingie was positioned far from the other breeds. Structure analysis revealed that the probable number of genetic clusters (K) was six and four with maximum likelihood and ΔK values, respectively. The clustering with maximum likelihood revealed that, in addition to the clustering of the other five breeds, the Satsumadori was subdivided into two genetic clusters. The clustering with ΔK value indicated that the broiler and Rhode Island Red were assigned to the same genetic cluster.

Characteristics of Seven Japanese Native Chicken Breeds Based on Egg White Protein Polymorphisms

【In this study, to examine genetic variability within a breed and genetic relationships between populations/breeds, we genotyped 606 birds from seven Japanese native chicken breeds at seven polymorphic loci of egg white proteins and compared those with Asian native chicken populations and commercial breeds. Genotyping of the Japanese native breeds showed that ovalbumin, two ovoglobulins and ovotransferrin were polymorphic, but ovomacroglobulin, ovoflavoprotein and lysozyme were monomorphic. The proportion of polymorphic loci ( $P_{poly}$ ) and average heterozygosity ( $\bar{H}$ ) within a population ranged from 0.286 to 0.429 and from 0.085 to 0.158, respectively. The coefficient of gene differentiation ( $G_{ST}$ ) was 0.250 in the Japanese native chicken breeds. This estimate was higher than that of Asian native chicken populations ( $G_{ST}$ = 0.083) 0.083) and of commercial breeds ( $G_{ST}$ = 0.169) 0.169). Dendrogram and PCA plot showed that Satsuma-dori, Jitokko, Amakusa-daio and Hinai-dori were closely related to each other and grouped into Asian native chickens and that Tsushima-jidori, Nagoya and Chan (Utaichan) were ramified far from other Japanese native chicken breeds. The egg white protein polymorphisms demonstrated that the population differentiation of the seven Japanese native chicken breeds was relatively large.】

Genetic mapping of quantitative trait loci affecting growth and carcass traits in F2 intercross chickens

We constructed a chicken F2 resource population to facilitate the genetic improvement of economically important traits, particularly growth and carcass traits. An F2 population comprising 240 chickens obtained by crossing a Shamo (lean, lightweight Japanese native breed) male and White Plymouth Rock breed (fat, heavyweight broiler) females was measured for BW, carcass weight (CW), abdominal fat weight (AFW), breast muscle weight (BMW), and thigh muscle weight (TMW) and was used for genome-wide linkage and QTL analysis, using a total of 240 microsatellite markers. A total of 14 QTL were detected at a 5% chromosome-wide level, and 7 QTL were significant at a 5% experiment-wide level for the traits evaluated in the F2 population. For growth traits, significant and suggestive QTL affecting BW (measured at 6 and 9 wk) and average daily gain were identified on similar regions of chromosomes 1 and 3. For carcass traits, the QTL effects on CW were detected on chromosomes 1 and 3, with the greatest F-ratio of 15.0 being obtained for CW on chromosome 3. Quantitative trait loci positions affecting BMW and TMW were not detected at the same loci as those detected for BMW percentage of CW and TMW percentage of CW. For AFW, QTL positions were detected at the same loci as those detected for AFW percentage of CW. The present study identified significant QTL affecting BW, CW, and AFW.

Evaluation of a reduction in the effective population size for a rare Japanese native chicken breed, the Kurekodori

Evaluation of a reduction in the effective population size for a rare Japanese native chicken breed, the Kurekodori

Genetic characterization of a Japanese native breed of chicken, the Hinai-dori, using microsatellite markers

Genetic characterization of a Japanese native breed of chicken, the Hinai-dori, using microsatellite markers

Genetic mapping of the QTL affecting body weight in chickens using a F 2 family

1. To identify the quantitative trait loci (QTL) affecting growth in chickens, we carried out QTL analysis on chicken growth traits using a population of 227 F 2 crosses between a Satsumadori (slow-growing, lightweight Japanese native breed used as a meat chicken) male and a White Plymouth Rock (early-maturing, heavy weight broiler). 2. We chose 78 microsatellite loci from 331 publicly available on 14 linkage groups, with respect to their utility and location. 3. Two QTLs affecting body weight at 13 and 16 weeks were mapped at 220 cM on chromosome 1 (LOD scores, 2.8 and 4.5, respectively, at 13 and 16 weeks), and at 60 cM on chromosome 2 (LOD scores, 6.2 and 8.1, respectively, at 13 and 16 weeks). 4. The closest loci to the QTLs were LEI71 on chromosome 1 and LMU13 and MCW184 on chromosome 2. 5. The sites of the QTLs agreed closely with those already reported. Therefore, it seems likely that QTLs affecting growth of chickens are located at these sites.

Genetic Mapping of the QTL Affecting Abdominal Fat Deposition in Chickens.

To identify the quantitative trait loci (QTL) affecting abdominal fat deposition in chickens, we carried out QTL analysis on this trait using a resource population of 222 F2 progeny from a cross between a Satsumadori (a Japanese native breed showing little abdominal fat deposition) male and a White Plymouth Rock (a broiler breed showing significant abdominal fat deposition) female. We chose 78 microsatellite loci from 331 loci publicly available of 14 linkage groups, based on their utility and location. One QTL affecting the ratio of abdominal fat deposition to live body weight (abdominal fat %) at 16 weeks of age was mapped at 38.0cM on chromosome 7, with a LOD score of 7.0. The closest loci to the QTL was MCW316. The QTL was located between markers MCW316 and MCW92, being 1.0cM apart from MCW316. The difference in abdominal fat % in F2 was associated with allele types of MCW316 (P<0.0005), which suggests the possibility of marker-assisted selection for abdominal fat deposition in chickens.

Genetic relationships among Japanese native breeds of chicken based on microsatellite DNA polymorphisms.

Genetic relationships among Japanese native breeds of chickens were studied on the basis of microsatellite DNA polymorphisms. DNA samples from 10 Japanese native breeds (Iwate-Jidori, Aizu-Jidori, Sadohige-Jidori, Siba-Tori, Onaga-Dori, Echigonankin, Hinai, Kinpa, Koeyoshi, and Tomaru) and one imported breed (White Leghorn) were analyzed using eight microsatellite markers that were isolated from a microsatellite DNA-enriched library of chickens (Takahashi et al. 1996). The PCR primers to detect (CA)n repeat length polymorphisms were synthesized based on the sequences of clones, and these markers were typed by PCR amplification and electrophoresis using a DNA sequencer. Since all eight microsatellite markers were polymorphic, genetic distance between the breeds could be calculated based on the frequencies of alleles of the microsatellites and phylogenetic relationships between the breeds could be estimated. Most Japanese native chickens were grouped into three groups that correspond to the origin breeds, Jidori, Shokoku, and Shamo. The results suggest that microsatellite DNA markers are a useful tool for studying the genetic relationships among chicken breeds.