Genetic Diversity of Wild Quail in China Ascertained with Microsatellite DNA Markers

The genetic diversity of domestic quail and two wild quail species, Japanese (Coturnix coturnix)and Common quail (Coturnix japonica), found in China was studied using microsatellite DNA markers. According to a comparison of the corresponding genetic indices in the three quail populations, such as Polymorphism Information Content (PIC), Mean Heterozygosity ( H ) and Fixation Index, wild Common quail possessed rich genetic diversity with 4.67 alleles per site. Its values for PIC and H were the highest, 0.5732 and 0.6621, respectively. Domestic quail had the lowest values, 0.5467 and 0.5933, respectively. Wild Japanese quail had little difference in genetic diversity from domestic quail. In addition, from analyses of the fuzzy cluster based on standard genetic distance, the similarity relationship matrix coefficient between wild Japanese quail and domestic quail was 0.937, and that between wild Common quail and domestic quail was 0.783. All of these results showed that the wild Japanese quail were closer to the domestic quail for phylogenetic relationship than wild Common quail. These results at the molecular level provide useful data about quail’s genetic background and further supported the hypothesis that the domestic quail originated from the wild Japanese quail. (


INTRODUCTION
Common quail (Coturnix coturnix) and Japanese quail (Coturnix japonica) belong to Aves, Calliformes, Phasianidae, Coturnix.The number of chromosomes is 78, including 6 big pairs, 6 medium pairs, and 27 mini pairs.Common quail includes European quail, African quail, and some kinds of Asian quail.Japanese quail are mainly found in East Asia, including Japan, Korea, China, Mongolia, Siberia, and Kuye Island, ranging from 100 to 150 degrees east longitude, and from 17 to 55 degrees north latitude (Sano et al., 1994).Most areas of China have the two wild species and the number of the Japanese quail is more than that of the Common quail.They are all migratory birds and their ranges greatly overlap.They usually inhabit the plains, coastal regions, and the foothills, especially places sparsely covered with grass.Domestic quail, derived from Japanese quail (Coturnix joponica), as laying, meat, and laboratory animals have produced a flourishing industry.At present, there are about 1,050 million quails around the world and 200 million quails in China.Quail ranks second to chicken in the Chinese poultry industry.Moreover, the use of quail is diversified including laying, meat, and laboratory types (Kimura, 1996).However, the existing domestic quails were all bred in Japan, and possess a narrow genetic background.After decades of use, the producing performance obviously does not satisfy the needs for the development of a flourishing industry.On the other hand, the deterioration of the environment in recent years dramatically reduced the number of wild quails; none could be found in some places.This paper analyzed the genetic diversity of the two wild quails with 9 microsatellite DNA markers, and compared them with domestic quails, aiming to determine genetic variance and phylogenetic relationship between wild and domestic quail populations in order to explore new wild quail resources and promote sustainable development of the quail industry.

Collection and extraction of genomic DNA
About 400 wild quail samples were captured from two abundant areas where wild quails migrated to and settled down.One was the Weishan Lake region, and the other was the Anyang district, Henan Province.One milliliter blood samples were collected from the heart of random samples of birds, including 40 wild Japanese quails (WSH) from the Weishan Lake region, 62 wild Common quails (YCQ) from the Anyang district, Henan Province, and 40 domestic quails (YJQ) from a quail breeding farm in Yangzhou City, Jiangsu Province.The genomic DNA was extracted according to the procedures described by Sambrook et al. (1998).DNA was extracted from 100 µl of whole EDTAblood.Then the mixture solution was made up with 100 µg/ml Proteinase K and 80 µg/ml Dnase-free pancreatic Rnase.After overnight incubation at 37°C, the proteins were removed by phenol and chloroform-isoamyl alcohol extractions and the DNA was precipitated by ethanol.

Source of primers
Nine primer pairs of microsatellite markers were designed according to the literature (Inoue-Murayama and Nomura, 1998;Inoue-Murayama et al., 2001;Kayang et al., 2000Kayang et al., , 2002;;Read et al., 2000), as shown in Table 1.Though 22 pairs of primers were tested in this work, only 9 pairs of primers were used in the analyses.

Polymerase chain reaction (PCR)
The PCR reaction mixture with a final volume of 25 µl contained 50 ng of template DNA, 2.5 µl of 10×buffer, 1.2 to 2.0 µl of 25 mmol/L MgCl 2 (as optimized for each marker), 0.5 µl of 10 mmol dNTP, 1 µl of 5 pmol/µl forward and reverse primers, and 1 u of Taq DNA Polymerase; ddH 2 O was added to the volume of 25 µl.
The amplification conditions for PCR were: 3 min denaturing at 94°C followed by 30 cycles of denaturation at 94°C for 1 min, annealing for 45 s at 38 to 58°C (as optimized for each marker), and extension at 72°C for 1 min.This was followed by a final cycle at 72°C for 15 min.The PCR products were then separated on 8% denaturing polyacrylamide gels with a molecular weight marker, pBR322 DNA/MspI Markers, on an electrophoresis system, at 100 V for 6 to 7 h, and stained with ethidium bromide.The results were visualized and photographed with the Kodak Digital Science ID Image Analysis System.
Test of H (Nei and Kimura, 2000) : For all polymorphic sites, the formula, , was used to calculate the difference in polymorphism between populations X and Y.
d and its variance, ) (d V were calculated using the following equations, where L' is the number of polymorphic sites.The difference in H between two populations was tested by the following formula, Fuzzy cluster analysis (Chang, 1998) : The following µ formula was used to analyze phylogenetic relationships between animal populations.Here J x ,J y and J x,y represent, respectively, population X, population Y, and the probability average among sites of the same alleles acquired at random from populations X and Y; According to the value of the membership function, the fuzzy resemblance matrix was set up, and finally a similarity relationship matrix was obtained through fuzzy operating.Fuzzy cluster analysis of different animal populations was based on the coefficients of the fuzzy similarity relationship matrix.

Calculation of allelic frequency
The electrophoresis results were determined by 8% denaturing polyacrylamide gels using a size marker, pBR322 DNA/MspI (Figure 1).Lanes 1, 3, and 6 represent heterozygotes, and lanes 2, 4, and 5 stand for homozygotes.According to the individual genotypes, the allelic frequencies of 9 microsatellite sites for the two wild quail populations and domestic population were calculated.A total of 37, 37, and 42 alleles in the WSH, YJQ, and YCQ populations were observed, respectively, which indicated that YCQ possessed richer genetic diversity than WSH and YJQ (Table 2).

Analysis of genetic variance of populations
Based on the allelic frequency of each site, PIC, H, mean H ( H ), N e and Fixation index were calculated (Tables 3 and 4).From Table 3, the value of PIC and H in YCQ was the highest being 0.5732 and 0.6621, respectively.Meanwhile, YJQ had the lowest values of 0.5467 and 0.5933, respectively.The difference in H presented the same trends, but there was no difference (p>0.05)among the three populations.N e of YCQ was 3.3913, and was higher than those of YJQ and WSH.This value of N e was close to the observed number per site of YCQ, 4.67.In the three quail populations, most values for the Fixation index were less than zero, except for GUJ0028 and GUQ0007 in YJQ and WSH, UBC0002 in YJQ, and GUQ0001 in YCQ.These results indicated that H in the three populations was high, namely there was no obviously deviation from the Hardy-Weinberg equilibrium.

Coefficient of gene differentiation (G st )
We can see that the G st value between YJQ and WSH was smaller than that between YJQ and YCQ (Table 6), which indicated that the extent of genetic differentiation between YJQ and WSH was lower, and these two species had a closer phylogenetic relationship compared to the other combinations.

Fuzzy cluster
The fuzzy similarity relationship matrix coefficients of fuzzy cluster of the three quail populations (Table 7) showed that WSH and YJQ firstly clustered together (Figure 2).The coefficient of fuzzy cluster matrix between them was 0.937, whereas the coefficient between YCQ and YJQ was 0.738.

Genetic analysis within two wild quail populations and a domestic population
PIC (Polymorphic Information Content), an important index of polymorphism of microsatellite DNA sites, was first used to estimate the polymorphism of gene markers in linkage analysis.The value of PIC indicates the degree of polymorphism.A site is highly polymorphic when PIC>0.5, normally polymorphic when PIC<0.5, and lowly polymorphic when PIC<0.25.Kong et al. (2006) assessed the genetic variation and established the relationship amongst breeds and strains using 15 chicken specific microsatellite markers, and founded that PIC of UMA1019 was the highest (0.872) and that of ADL0234 was the lowest (0.562).Tu et al. (2006) studied genetic diversity of 14 indigenous grey goose breeds in China based on microsatellite markers, and indicated the highest PIC was in the Xupu (0.6916) and the lowest was in the Yan (0.4985).
In contrast to that of chicken and other fowls, quail's microsatellite DNA has not received much attention from researchers.Though few highly polymorphic microsatellite DNA sites of quail were reported, there were 5 highly and 4 normally polymorphic sites in YJQ in this study.WSH had 5 highly polymorphic, 3 normally polymorphic, and 1 lowly polymorphic site, and in the YCQ population, there were 7 highly polymorphic, 1 normally polymorphic, and 1 monomorphic site, which showed obvious differences from the two other populations.Heterozygosity, a measure of gene diversity, reflects the genetic variance of populations at polymorphic sites.The GUJ0028, GUJ0029, UBC0004, UBC0005, and UBC0006 sites possessed high polymorphism, rich genetic diversity, and high selection potentials (Table 3).Therefore, they may act as candidate genes to be applied to the study of location of QTL.Liu et al. (2006) studied the correlations between heterozygosity at microsatellite loci, mean d2 and body weight in a Chinese native chicken, and indicated positive correlations were found between microsatellite heterozygosity and body weight in males and females (p<0.05).The difference in genetic differentiation level between YJQ and WSH was lower, though the test of H showed no significant differences among the three quail populations (p>0.05).Furthermore, N e showed the same trends as H.It is well known that, the more similar the distribution of alleles in populations, the more close the relation between N e and the absolute number of alleles tested, which is the reciprocal of homozygosity (Nozawa et al., 1996).Of the 9 microsatellite sites in this paper, the N e value of GUQ0001 and GUQ0007 in their own populations was relatively small, indicating that the distribution of alleles was not similar.However, the condition of UBC0006 and GUJ0029 was the opposite.
At most sites the Fixation Index (F) was negative (Table 4), showing that the heterozygote frequencies were high at these sites.The heterozygote of YCQ was relatively richer than that of YJQ and WSH.However, there existed no significant genetic differentiation and evolutionary divergence force in the three populations.
Through comparison of domestic quail and the two wild quails, we found that the genetic variance level of WSH was close to that of YJQ.The possible reasons for this result include common origin and evolutionary progress, time of species formation, bottleneck effect at the beginning of domestication, gene flow between different species, crosses between different lines, mixing, and human activities.

Genetic analysis of three quail populations
The genetic background could be evaluated through the allele composition of the three quail populations.Thirtyseven alleles were detected in YJQ and WSH at 9 microsatellite sites, with a mean of 4.11 alleles per site.Moreover, every allele was shared by the two populations, and the distribution of alleles in YJQ and WSH was still under equilibrium due to geographical isolation and artificial selection.On the other hand, 42 alleles were detected in YCQ at the same 9 microsatellite sites, with a mean of 4.67 alleles per site.However, there was one homogeneous site in YCQ, which presented a difference from the two other populations, YJQ and WSH.
G st is the right index of calculating relative value of genetic differentiation between sub-populations.Chang et al. (2000) reported that the G st of a goat population in the middle and lower Yellow River in China was 0.0038-0.2118.Fan et al. (1999) tested G st of 8 native pig breeds in China, and found a value of 0.2030.In this study, G st of 3 quail populations was 0.0109-0.0548;that is, the genetic variance between populations accounted for 1.09%-5.48% of the total genetic variance, which indicated that the variance was mainly produced from within populations.
Recently, there have been many studies of phylogenetic relationship in animal populations with standard genetic distance and classical cluster.Zhang et al. (1998) analyzed the population genetic variance in Guangdong local chicken breeds and clarified the phylogenetic relationship among these breeds.Cao et al. (1999) studied the genetic variance of 5 beef populations with microsatellite markers.Wu et al. (2004) reported the genetic structure of 12 local chicken breeds in China with the microsatellite technique and divided the 12 breeds into 3 clones based on their phylogenetic relationships.Osman et al. (2006) also revealed the genetic variability and relationships of Japanese and foreign chickens assessed by microsatellite DNA profiling and indicated native Japanese chicken breeds and foreign breeds were clearly separated from each other.Olowofeso et al. (2006) also studied genetic distance of the four chicken populations and indicated the Jiangchun and Cshiqishi chickens were closely related breeds.Su et al. (2006) reported the genetic variance of different Chinese duck populations and presented the genetic diversity was improved by crossbreeding.When it came to quail, few reports about phylogenetic relationship were available.Moreover, most reports about the variance were based on allied species with cytogenetic and biochemical genetic techniques, and the application of the theory of Nei's standard genetic distance to analyze phylogenetic relationships.It was obvious that this theory had the trend of absolution, overlooking the consistency of difference between species or breeds and did not show the relativity of the results.It belonged to hard clustering (Chang et al., 2001).Fuzzy cluster analysis, combining the characteristics of animal genetics into fuzzy set theory, better fits the objective facts.This paper changed the coefficients of Nei's standard genetic distance into membership functions, and then set up the fuzzy resemblance matrix, after fuzzy operating step by step.Lastly a similarity relationship matrix was obtained.According to the coefficients of the fuzzy similarity relationship matrix, we got the fuzzy clustering of the three quail populations (Figure 2).The figure showed that the difference in genetic diversity between YJQ and WSH was smaller than that between YJQ and YCQ, and clustered at the level of 0.937.The results further proved that YJQ and WSH were closer in their phylogenetic relationship than YJQ and YCQ.There would be a reasonably deduced common origin and evolutionary progress in fairly recent times between YJQ and WSH.
The mysteries concerning the origin and domestication of quail Presently, academic circles tend to believe that the domestic quail originated from wild quail in East Asia, but whether the origin of domestic quail is from the Japanese Islands, China, or Korea is a controversial issue.Recent studies showed that the wild quail populations of the Japanese Islands cluster alone in the phylogenetic clustering with domestic populations of different parts of the world.This indicates that the phylogenetic relationship of domestic quail is distant from that of wild quail in the Japanese Islands (Chang et al., 2001).According to the literature, it has been 1,000 years since China began to domesticate quail, 400 years earlier than Japan.China and Japan are separated only by a strip of water.Cultural exchange between the two countries has a long history.During the period that the Sui and Tang Dynasties (581 A.D.-907 A.D.) ruled China, with the return of Japanese students studying abroad, envoys to the Sui and Tang Dynasties, and learned monks, the custom of quail-fighting spread from China to Japan; so, Chinese quail culture had some effect on that of Japan.This historical background shows the possibility that domestic quail originated from WSH in China.In addition, recent reports proposed that WSH and domestic quail populations have a closer phylogenetic relationship than with the Japanese wild quail based on the analysis of enzyme polymorphisms (Chang et al., 2005).This paper further supported on the DNA level that domestic quail may originate from WSH.All of the alleles at 9 microsatellite sites were shared by WSH and YJQ, which provided an objective foundation for determining the origin of quail and the location of early domestication.
the fuzzy consistency relationship matrix R between populations X and Y.

Table 1 .
Characterization of the nine microsatellite sites and PCR conditions

Table 4 .
Effective number of alleles (N e ), observed number of alleles (N o ), and Fixation index (F) of nine microsatellite DNA sites in three quail populations

Table 7 .
Fuzzy similarity relationship matrix coefficients of fuzzy cluster of Domestic quail (YJQ), wild Japanese quail (WSH) and