| The level of genetic differentiation and genetic structure in Bian chicken of 0 generation and two controlled chicken populations (Jinghai chicken and Youxi chicken) were analysed based on 29 microsatellite markers; In order to see the effect of conservation in Bian chicken, twenty-one microsatellite markers which had 4 alleles in Bian chicken of 0 generation were choosen to analyse the diversity of Bian chicken in 1 generation and compared the diversity with Bian chicken of 0 generation; PCR-SSCP method was used to detect the SNPs of the Myostatin Gene in Bian chicken, using Jinghai chicken, Youxi chicken and Arbor Acre chicken as controlled populations. We analysed its relationship with growth traits and reproductive traits in Bian chicken as well in order to lay the foundation for Marker-assisted Selection (MAS) for Bian chicken; In order to find out the molecule mechanism that the mutation G2283A had significant effect both on growth traits and reproductive traits, FQ-PCR method was used to study the expreesion level in breast muscle, leg muscle and ovary among 3 genotypes which were formed by the mutation G2283A in the Myostatin gene. The expreesion level in breast muscle, leg muscle and ovary in Bian chicken was also studied. The main results are listed as follows:1. Based on 29 microsatellite markers, a total of 166 distinct alleles were observed across the 3 breeds (Bian chicken, Jinghai chicken and Youxi chicken), and 32 of these alleles (19.3%) were unique to only 1 breed. Bian chicken carried the largest number of private alleles at 15 (46.9%), followed by the Jinghai chicken with 12 private alleles (37.5%). The average polymorphism information content (0.5168) and the average expected heterozygote frequency (0.5750) of the Bian chicken were the highest, and those of the Jinghai chicken were 0.4915 and 0.5505, respectively, which were the lowest. Among 29 microsatellite loci, there were 15 highly informative loci in Bian chicken, and the other 14 were reasonably informative loci. The highly informative loci in Jinghai chicken and Youxi chicken were 17 and 14 respectively. Significant deviations from the Hardy-Weinberg equilibrium were observed at several locus-breed combinations, showing a deficit of heterozygotes in many cases. As a whole, genetic differentiation among the breeds estimated by the fixation index (Fst) were at 6.7% (p<0.001). The heterozygote deficit within population (Fis) was 22.2% (p<0.001), with the highest (0.249) in Bian chicken and lowest (0.159) in Youxi chicken. These results serve as an initial step in the plan for genetic characterization and conservation of the chicken genetic resource of Bian, as well as Jinghai and Youxi chickens.2. Twenty-one microsatellite markers which had 4 alleles were choosen to analyse the diversity of Bian chicken in the two generations. The results showed that a total of 122 alleles were detected, of which, 102 were detected in the two generations, and 9 alleles were unique to generation 0. The average polymorphism information content (PIC) of generations 0 and 1 was 0.5473 and 0.5437, respectively, and the average expected heterozygote frequency (He) of the two generations was 0.5967 and 0.6009, respectively. The inbreeding coefficient was 0.233 in generation 0 and 0.134 in generation 1. Through selection and breeding for one generation, the PIC and He were maintained, while the inbreeding coefficient (Fis) was somewhat decreased. It indicated that the conserved method was feasible. The conservation method not only conserved the genetic diversity of Bian chicken, but also avoided inbreeding depression.3. Seventeen mutations were detected in the four chicken breeds. Amongst them, 10 mutations (A326G, C334G, G673A, G985C, G1085A, A1278T, C1346T, G1375A, A1473G and G1491A) were located in the 5′regulatory region of the Myostatin gene and 4 mutations (G2100A,G2109A,C2244G, G2283A) were located in exon 1. The remaining 3 mutations (C7552T, C7638T, T7661A) were located in exon 3 . Mutations in the coding region of exons were synonymous mutations.4. In Bian chicken, Jinghai chicken, Youxi chicken and AA chicken there were 7, 9, 9 and 5 loci displayed polymorphisms in the Myostatin gene respectively. MYOE1-2 in Bian Chicken was a highly informative locus. MYO1 and MYOE1-3 were reasonably informative loci. The remaing 4 were slightly informative loci. The highly informative loci in Jianghai Chicken, Youxi Chicken and AA Chicken were 1, 2 and 0 respectively. The reasonably informative loci were all 4 in Jianghai Chicken, Youxi Chicken and AA Chicken .5. Through online software prediction it showed that the mutations in 5′regulatory region of the Myostatin gene in 4 chicken breeds resulted in 10 transcription factor binding site (E2F, CRE-BP, CREB, HFH-2, NKx-2, CdxA, Oct-1, V-Maf, Tst-1 and C/EBPα) changed.6. MYO1, MYO6 and MYO7 in the 5′regulatory region of the Myostatin gene and MYOE1-2, MYOE1-3 in the exonic region had significant effect on growth traits (P<0.05 or P<0.01). It suggested that Myostatin gene could be the major gene or is linked to the major gene that affects the growth traits in Bian chicken. For primers MYOE1-3, Bian chicken with genotype DE and EE had larger body weight than those with genotype DD (P<0.05 or P<0.01) from 6 to 18 wk of age. MYOE1-3 had a great potential to be used as genetic marker for growth traits in Bian chicken.7. MYO1, MYO6 and MYO7 in the 5′regulatory region of the Myostatin gene had significant effect on body weight at first egg (P<0.05). MYOE1-2, MYOE1-3, MYOE3-2 and MYOE3-3 in the exonic region had significant effect on age at first egg and egg number at 300 days (P<0.05 or P<0.01). It suggested that the Myostatin gene could be the major gene or is linked to the major gene that affects the reproductive traits in Bian chicken. For primers MYOE1-3, Bian chicken with genotype DD had larger age at first egg than those with genotype DE and genotype EE (P<0.05 and P<0.01 respectively). Correspondingly, Bian chicken with genotype EE and DE had larger egg number at 300 days than those with genotype DD (P<0.01 and P<0.05 respectively). MYOE1-3 had a great potential to be used as genetic marker for egg number at 300 days in Bian chicken.8. Significant interaction effect between different loci of the Myostatin gene were found on growth traits and reproductive traits (P<0.05 or P<0.01) in Bian Chicken.9. Using FQ-PCR method, it showed that the expression level was differ in breast muscle and leg muscle among 3 genotypes which were formed by the mutation G2283A in the Myostatin gene, while there was no difference in ovary among the 3 genotypes. In breast muscle, the expression level of genotype DD was 1.40 times than genotype DE and 4.66 times than genotype EE. In leg muscle, the expression level of genotype DD was 1.27 times than genotype DE and 8 times than genotype EE. The results also showed that Myostatin is principally expressed in breast muscle and leg muscle, while the expression level was very low in ovary. The expression level in breast muscle and leg muscle was 471.1 and 501.5 times than that in ovary respectively.
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