MATERIALS AND METHODS

Samples: Yangzhou (n = 71), Sichuan (n = 111), Rhine (n = 45), Landaise (n = 37), White Roman (n = 69) and Wanxi (n = 35) from 6 geese breeding farms of Anhui province (Anhui, China) were selected in 2009 and 2010. Genomic DNA from goose wing’s blood was extracted by phenol-chloroform method (Sambrook and Russell, 2001) and then dissolved in TE buffer [10 mmol L^-1 Tris-HCl (pH 8.0), 1 mmol L^-1 EDTA (pH 8.0)] and kept at -20°C.

The grade of egg productivity (GEP) of six goose breeds were estimated according to the practical egg-yield and less than 20 eggs per year as level 1, then each additional 15 eggs to improve a grade, respectively.

Primers: Based on the sequence of the magang goose PRL gene (GenBank DQ345782.1), a pair of primers was designed using the primer premier 5.0 program (http://www.premierbiosoft.com/) to amplify the fragment containing 5’ partial regulatory region, 5’-UTR, exon 1 and partial intron 1 of goose PRL gene. The sequence length of PCR product was 407 bp in which fragment length (PRL268) of 5’ regulatory region and 5’-UTR was 268 bp. The primers were synthesized by Shanghai Bioengineering Inc. (Shanghai, China).

PCR conditions and procedures: PCR was carried out using 25 μL PCR amplification kits: 1 μL genomic DNA (50 ng μL^-1), 2.5 μL of 10 x buffer with Mg²⁺, 2 μL dNTP (2 mM), 0.5 μL (2.5 units μL^-1) Taq DNA polymerase (Beijing Dingguo Biotechnology Inc. China), 0.5 μL of each primer and 18 μL double-distilled (dd) H₂O. Amplification conditions were: initial denaturation of 5 min at 94°C; 35 cycles of 94°C for 30 sec, 56°C for 30 sec, 72°C for 50 sec, followed by 72°C extension for 10 min. The PCR products were detected using 1.5% agarose gel electrophoresis.

Polymorphism analysis: The PCR products digested with BstYI endonucleases (MBI) and then electrophoresed on 3% agarose gel for genotyping polymorphisms. The gene frequencies of the different goose breeds were analyzed statistically. Allele gene frequency, p and q, was calculated by p = D + 0.5H and q = 1-p, respectively where D and H represent homozygote and heterozygote gene frequency, respectively. The distribution patterns of different genotypes were analyzed in the six breeds.

Dual-reporter gene analysis: According to Chen’s protocol (Chen, 2010), the transcriptional efficacy of luciferase gene with different PRL268 promoters in 293T cells were tested and analyzed using dual-reporter gene assays.

Statistical analysis: The PRL268 fragment was subjected to promoter prediction in BDGP (Reese, 2000, 2001). The ratio of firefly luciferase to renilla luciferin intensity was calculated. For the data of non-normal distribution, the values need be transformed using logarithmic transformation (Olivier et al., 2008) and evaluated for p = 0.05 significant differences between different promoters using student’s t-test.

RESULTS

Based on the sequencing results of mixed DNA samples, a mutation locus was detected and located at 10 bp upstream of the CDS start-point (G-10T). Incision of the PCR product by BstYI revealed three genotypes: TT (384/23bp), TG (384/235/149/23bp) and GG (235/149/23bp). The sequence of PRL268 is shown in Fig. 1.

The allele types of G-10T in different breeds were shown in Table 1. The G allele frequencies in Rhine, White Roman and Landaise were all above 0.90 and reached 0.7857 in Wanxi. However, the G allele frequencies in Yangzhou and Sichuan were only 0.1479 and 0.2117, respectively. Hardy-weinberg equilibrium tests (Zhou et al., 2009) and population genetic structure are shown in Table 2. The heterozygosities of Wanxi, Sichuan and Yangzhou were 0.3367, 0.3338 and 0.2520 and their polymorphic information contents were 0.2800, 0.2781 and 0.2203, respectively which showed higher genetic polymorphism comparing with that of Rhine, White Roman and Landaise. The six goose populations all were in Hardy-weinberg equilibrium.

From Table 1, GEPs of Rhine, White Roman, Landaise and Wanxi were listed as lower grade group and higher G allele frequencies, while Yangzhou and Sichuan were referred to as higher grade group and higher T allele frequencies, indicating that the T allele frequency distribution was closely associated with higher egg productivity (Fig. 2). T allele frequencies of higher and lower grade groups were 0.8132 and 0.0833, respectively.

Fig. 1:	The sequence and characters of the PRL268 fragment. The italics and bold letters are TATA-box. The capital letters are 5’-UTR of mRNA of prolactin gene. The g/t and G/U are mutation loci. The capital and italics letters are coding region

Table 1:	G-10T allele frequencies and grade of egg productivity

Table 2:	Hardy-Weinberg equilibrium tests and population genetic structure

Fig. 2:	Gene frequency distribution in goose breeds with grade of egg productivity (GEP). Values represent T allele frequency

Table 3:	Promoter efficiency of PRL268 using dual reporter gene assays
*Significant difference between means: A>B>C, p<0.01

Table 3 showed the efficacy of PRL268 with T or G allele as promoter of the luciferase reporter gene (PRL268T or PRL268G) in eukaryotic cells. The results shown the promoter efficacy of PRL268G was 5.5140 and increased 26.14% than that of PRL268T (p<0.01), indicating that the T allele affected the transcriptional efficacy of the luciferase reporter gene.

DISCUSSION

There are several reports about the correlation of PRL genotype with avian nesting behavior and egg productivity (Reddy et al., 2006b; Jiang et al., 2009; Eltayeb et al., 2010). A well-known hypothesis is that reduced PRL expression will increase egg productivity. This is consistent with the results that are the Wanxi with higher broodiness and lower GEP in the six goose breeds, followed by the Landaise, White Roman and Rhine with moderate broodiness and GEP and the Yangzhou and Sichuan with the weakest broodiness and higher GEP. However, the linkage between broodiness, egg productivity and PRL gene modulation remains unclear. For a long time, the identifying of the major gene on egg productivity is limited by the population size available (Zou, 2009; Kaiser et al., 2003). Actually, differences in reproductive performance among populations have been reflected in gene frequency via selection pressure. The correlation between GEP and the distribution of PRL gene polymorphisms in six goose breeds showed a consistency in role of PRL gene, although other genes might also have similar effects. In the present study, the distribution pattern of G-10T reflected real differences in GEP. The geese with T allele had higher GEP whereas the geese with G allele exhibited the lower GEP. From the Table 3, the promoter efficacy of PRL268G increased significantly 26.14% than that of PRL268T which showed that T allele might have the role of down regulated transcription of PRL gene while G allele might play the up regulated role. This may be of use in marker assisted selection for the genetic improvement and breed resources protection in goose egg productivity.

Genetic mechanism of T-10G on PRL level and GEP is not clear. The sequence (T/G)ATCTC motif of T-10G locus may serve as the binding of the some transcription factors (Fu and Marzluf, 1990) or ribosomes recognition element in mRNA (Tuller et al., 2009; Baranick et al., 2008). On the other hand, the sequence CAGUAUCUCUACC in 5'-UTR of T allele may produce a potential miRNA targeting locus that is similar to the chicken gga-miR-1688 (Glazov et al., 2008) and then affect the translation of mRNA for PRL. These reviews are in support to the mechanism of T-10G as a marker locus for PRL level and GEP.

CONCLUSION

According to the result of this study, G-10T of prolactin gene is a SNP locus affecting serum PRL level of geese. The T allele frequency is higher in goose breeds with higher GEP. G-10T locus may be associated with egg productivity and broodiness of goose breeds.

ACKNOWLEDGMENTS

The work was supported by The National Key Technology R and D Program of China (2008BADB2B01) and Major Projects of New Varieties of Genetically Modified Organisms of the Ministry of Agriculture (2009ZX08008-007B).