R. Tadano

High genetic divergence in miniature breeds of Japanese native chickens compared to Red Junglefowl, as revealed by microsatellite analysis

A wide diversity of domesticated chicken breeds exist due to artificial selection on the basis of human interests. Miniature variants (bantams) are eminently illustrative of the large changes from ancestral junglefowls. In this report, the genetic characterization of seven Japanese miniature chicken breeds and varieties, together with institute-kept Red Junglefowl, was conducted by means of typing 40 microsatellites located on 21 autosomes. We drew focus to genetic differentiation between the miniature chicken breeds and Red Junglefowl in particular. A total of 305 alleles were identified: 27 of these alleles (8.9%) were unique to the Red Junglefowl with high frequencies (>20%). Significantly high genetic differences (F(ST)) were obtained between Red Junglefowl and all other breeds with a range of 0.3901-0.5128. Individual clustering (constructed from combinations of the proportion of shared alleles and the neighbour-joining method) indicated high genetic divergence among breeds including Red Junglefowl. There were also individual assignments on the basis of the Bayesian and distance-based approaches. The microsatellite differences in the miniature chicken breeds compared to the presumed wild ancestor reflected the phenotypic diversity among them, indicating that each of these miniature chicken breeds is a unique gene pool.

High accuracy of genetic discrimination among chicken lines obtained through an individual assignment test

In the current study, we carried out assignment tests applying the Bayesian and distance-based methods, using 20 microsatellite genotypes in four chicken lines. The Bayesian method showed slightly higher performance of assignment than the distance-based method. In the assignment using the Bayesian method, >or=90% accuracy of assignment was attained by using only two of the most heterozygous markers, whereas in the case of the least heterozygous markers, six were needed to reach the same level of accuracy. In the assignment of the most closely related line pair (F(ST) = 0.1736), at least 12 markers selected by random ordering and at least 15 individuals per line were needed to stably obtain high accuracy of assignment (>or=97%), whereas using only six random markers achieved 97-100% of accuracy between the two most distinct lines (F(ST) = 0.3651) without reference to the sample size per line.

A genetically female brain is required for a regular reproductive cycle in chicken brain chimeras

Sexual differentiation leads to structural and behavioural differences between males and females. Here we investigate the intrinsic sex identity of the brain by constructing chicken chimeras in which the brain primordium is switched between male and female identities before gonadal development. We find that the female chimeras with male brains display delayed sexual maturation and irregular oviposition cycles, although their behaviour, plasma concentrations of sex steroids and luteinizing hormone levels are normal. The male chimeras with female brains show phenotypes similar to typical cocks. In the perinatal period, oestrogen concentrations in the genetically male brain are higher than those in the genetically female brain. Our study demonstrates that male brain cells retain male sex identity and do not differentiate into female cells to drive the normal oestrous cycle, even when situated in the female hormonal milieu. This is clear evidence for a sex-specific feature that develops independent of gonadal steroids.

Analysis of genetic divergence between closely related lines of chickens

The aim of the present study was to characterize genetic diversity within and differentiation between 5 closely related lines of Japanese-native chickens, the Nagoya breed, based on microsatellite polymorphisms. For 5 Nagoya lines, the mean number of alleles per locus, the observed heterozygosity, the expected heterozygosity, and the inbreeding coefficient within a line ranged from 2.35 to 2.85, from 0.385 to 0.507 (average = 0.438), from 0.404 to 0.480 (average = 0.433), and from -0.056 to 0.074, respectively. These results indicated that Nagoya lines have moderate levels of genetic diversity and no severe inbreeding signatures. Genetic differentiations between pairs of lines (pairwise F(ST)) ranged from 0.0224 to 0.2500. The lowest differentiation was found between 2 lines that were divided into distinct lines about 10 years ago. Genetic clustering analyses, the neighbor-joining tree constructed from genetic distances of the proportion of shared alleles and the Bayesian model-based clustering, were carried out based on multilocus genotypes of individuals. The results suggested that Nagoya lines were genetically distinct from commercial gene pools (broilers and white- and brown-egg layers) and that the Nagoya breed is a unique genetic resource. The results from the present study have the potential to contribute to future breeding and management of lines of the Nagoya breed.

Genetic structure and differentiation of the Japanese extremely long-tailed chicken breed (Onagadori), associated with plumage colour variation: suggestions for its management and conservation

The Onagadori is a distinguished chicken breed that is characterized by an extremely long tail in the male. In this breed, three different plumage colour varieties have been developed (black-breasted white, black-breasted red and white) in which the black-breasted white is believed to be the original colour of the Onagadori, based on historical records. To establish a conservation strategy, 176 birds were genotyped for autosomal microsatellites. Significant genetic distinctness was found between the original (black-breasted white) and two derivative varieties (F(ST) = 0.091 and 0.093). At the same time, a Bayesian model-based clustering revealed that the majority of individuals belonging to the black-breasted red and white varieties had an extremely low proportion of the genome shared with the original type (black-breasted white). This suggests that derivative varieties were created by crossing with other breeds, with low introgression of the original-type genome. We propose that the three plumage colour varieties should be treated as separate genetic units in a conservation programme.

Cost-effective development of highly polymorphic microsatellite in Japanese quail facilitated by next-generation sequencing.

Next-generation sequencing technologies permit rapid and cost-effective identification of numerous putative microsatellite loci. Here, from the genome sequences of Japanese quail, we developed microsatellite markers containing dinucleotide repeats and employed these for characterisation of genetic diversity and population structure. A total of 385 individuals from 12 experimental and one wild-derived Japanese quail lines were genotyped with newly developed autosomal markers. The maximum number of alleles, expected heterozygosity and polymorphic information content (PIC) per locus were 10, 0.80 and 0.77 respectively. Approximately half of the markers were highly informative (PIC ≥ 0.50). The mean number of alleles per locus and observed heterozygosity within a line were in the range of 1.3-4.1 and 0.11-0.53 respectively. Compared with the wild-derived line, genetic diversity levels were low in the experimental lines. Genetic differentiation (FST ) between all pairs of the lines ranged from 0.13 to 0.83. Genetic clustering analyses based on multilocus genotypes of individuals showed that most individuals formed clearly defined clusters corresponding to the origins of the lines. These results suggest that Japanese quail experimental lines are highly structured. Microsatellite markers developed in this study may be effective for future genetic studies of Japanese quail.

Genetic differentiation among White Leghorn lines: Application of individual-based clustering approaches

Genetic differentiations among White Leghorn lines were quantified based on allele frequencies of 40 microsatellite loci. In the survey among 7 lines, a considerable degree of differentiation was estimated between each pair of lines; genetic differentiation index (pairwise F(ST)) ranged from 0.0706 to 0.2590. Furthermore, 2 genetic clustering analyses of individuals, a neighbor-joining approach based on interindividual distances and the Bayesian procedure, which can assign individuals to the origins of their lines based on information on multilocus genotypes, were applied to a pairwise comparison of line differentiation. In the clustering approaches between the lowest differentiated line pair (pairwise F(ST) = 0.0706), individuals from 2 different line origins could not be separated into 2 distinct clusters, which indicates that the genetic boundary of these lines is ambiguous. On the other hand, between the highest differentiated pair (pairwise F(ST) = 0.2590), all individuals could be strictly clustered into 2 distinct groups, consistent with the origins of their lines. In the clustering based on interindividual distances, firm separations of individuals were observed in only relatively highly differentiated pairs of lines. Furthermore, in the Bayesian procedure, even in pairs with a relatively low differentiation, individuals from 2 lines formed 2 distinct clusters according to their origins. The results of the present study suggest that chicken lines possess considerable genetic differentiation despite their common breed origin. These clustering approaches at the individual level may be useful for the genetic identification and characterization of poultry stocks.

Comparison of microsatellite variations between Red Junglefowl and a commercial chicken gene pool

It is assumed that Red Junglefowl (Gallus gallus) is one of the main ancestors of domestic chickens (Gallus gallus domesticus). Differences in microsatellite polymorphisms between Red Junglefowl and modern commercial chickens, which are used for egg and meat production, have not been fully reported. A total of 361 individuals from 1 Red Junglefowl population that has been maintained as a closed flock, 5 final cross-bred commercial layer populations (white-, tinted-, and brown-egg layers), and 2 final cross-bred commercial broiler populations were genotyped for 40 autosomal microsatellite loci. We compared microsatellite variations in Red Junglefowl with those in a commercial chicken gene pool. The contribution of each population to the genetic diversity was also estimated based on the molecular coancestry. In total, 302 distinct alleles were detected in 1 Red Junglefowl and 7 commercial chicken populations, of which 31 alleles (10.3%) were unique to Red Junglefowl, most of which occurred at a high frequency. The genetic differentiation between Red Junglefowl and commercial chickens (pairwise FST) ranged from 0.32 to 0.47. According to the neighbor-joining tree based on the modified Cavalli-Sforza chord distances and the Bayesian clustering analysis, Red Junglefowl was genetically distant from the commercial chicken gene pool tested. In all of the populations analyzed, Red Junglefowl made the highest contribution to genetic diversity. These results suggest that Red Junglefowl has a distinct distribution of microsatellite alleles and that there is a high level of genetic divergence between Red Junglefowl and commercial chickens.

Genetic characterization and conservation priorities of chicken lines

Molecular markers are a useful tool for evaluating genetic diversity of chicken genetic resources. Seven chicken lines derived from the Plymouth Rock breed were genotyped using 40 microsatellite markers to quantify genetic differentiation and assess conservation priorities for the lines. Genetic differentiation between pairs of the lines (pairwise FST) ranged from 0.201 to 0.422. A neighbor-joining tree of individuals, based on the proportion of shared alleles, formed clearly defined clusters corresponding to the origins of the lines. In Bayesian model-based clustering, most individuals were clearly assigned to single clusters according to line origin and showed no admixture. These results indicated that a substantial degree of genetic differentiation exists among the lines. To decide priorities for conservation, the contribution of each line to the genetic diversity was estimated. The result indicated that a loss of 4 of the 7 lines would lead to a loss from 1.14 to 3.44% of total genetic diversity. The most preferred line for conservation purposes was identified based on multilocus microsatellite analysis. Our results confirmed that characterization by means of molecular markers is helpful for establishing a plan for conservation of chicken genetic resources.

Genetic Differentiation among Commercial Lines of Laying-type Japanese Quail

Recently in Japan, approximately six million quails were primarily being reared for commercial egg production. It is believed that almost all commercial quails in the country became extinct during World War II, and that the present commercial gene pool was restored from the limited number of surviving birds. The present study evaluates the genetic diversity within and differentiation between 12 laying-type Japanese quail lines on the basis of 45 microsatellite genotypes. The mean number of alleles per locus and the expected heterozygosity within a quail line were 5.22–5.69 and 0.601–0.618, respectively. These results showed that laying-type quail lines in the present study exhibited a higher degree of genetic diversity than experimental quail lines in a previous study. Pairwise genetic differentiations (FST) between lines were significant but weak (FST=0.0028–0.0254; 57.6%), and no significant differentiations were found between the remainder. This was also confirmed by genetic clustering analyses, in which individuals did not form independent clusters consistent with their line origins. The results of the present study indicate relatively high genetic diversity within and no clear genetic differentiation between laying-type quail lines. Absence of genetic differentiation may reflect the breeding history of laying-type quails.