Abstract: In this study, the exons polymorphism of Growth Hormone (GH) gene were detected by Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) and DNA sequencing method in 109 individuals from Shitou goose, Wanxi white goose and Zi goose. About 5 pairs of primer were designed according to the GH gene sequence of goose then the association of polymorphism of GH gene with the growth traits was studied. The results indicated there were two nucleotide mutations at P2 and P4 locus, respectively which resulted in 10 genotypes and 3 genotypes. The χ²-test showed that the genotype distributions of GH gene were in agreement with Hardy-Weinberg equilibrium in P2 and P4 locus (p>0.05), the difference was extremely significant in P2 locus on the distribution of genotype for Shitou goose and Zigoose while for Shitou goose, Wanxi white goose and Zi goose, it was extreme significant at P4 locus but there was no difference for the other locus. The least square on genotypes and groups analysis showed that there were significant differences (p<0.01) on growth traits in different groups however, only P4 locus have genotypes effect, the average value of AA genotype was the highest therefore, AA genotype could be the favorable marker for early breeding selection for high product trait of local goose.

INTRODUCTION

So far, the genomic structure of the GH gene has been studied in chicken, duck and goose. These animals share a similar gene structure containing 5 exons and 4 introns. GH gene polymorphisms have also been observed in other poultry and animals.

A recent study on dairy cattle demonstrated that an MspI polymorphism at intron 3 of bovine GH gene was linked to the content of milk protein. About 2 polymorphic sites were found in intron 2 of Laiyin goose GH gene and were significant differences on egg-layingtest. Studies of genetic polymorphisms at the nucleotide level brought a promising way to improve growth traits, especially by making conventional breeding much more powerful and efficient. This will be achieved primarily by making possible the identification at birth of interesting genotypes before performance recording and incorporation of marker information in conventional selection.

Therefore, this study used PCR-SSCP method to detect genetic variation of all exon of GH gene in three local goose (Shitou goose, Wanxi White goose and Zi goose) and the aim of the present study was to show the effect of body weight on growth traits and to investigate genes that are potentially associated with this changes.

MATERIALS AND METHODS

Zi goose (N = 50). Wan-xi White Goose (N = 59) and shitou (N = 64) goose was obtained from Gene Pool of National Waterfowl (Taizhou) and reared under the same environment and management.

At 10 weeks of age, the blood samples were taken from the wing vein, heparin sodium was used as an anticoagulant. Genomic DNA utilized in PCR amplification was extracted by routing phenol-chloroform method. The rTaq polymerase, dNTP, primer were from Sangon Bioengineering (Shanghai) company and the others were from Baosheng Bioengineering (Dalian) Co. Ltd.

Primers design: About 5 primers were designed and conducted PCR according to the GH gene sequence of duck in Gen bank database (accession number AB158760), the primer sequence and PCR product size was shown in Table 1.

PCR amplification and SSCP analysis: PCR reactions were performed in (Eppendorff) according to following program: initial denaturation for 5 min at 94°C and then 34 cycles (94°C 30 sec; 51-62°C 30 sec; 72°C 50 sec) and final extension for 10 min at 72°C. The PCR reaction mix in a total volume of 20 μL contained: 10xPCR reaction buffer (including Mg²⁺), 1.0 μL of dNTP mix (2.5 mM), 0.2 μL of Taq DNA polymerase (5 U μL^-1), 0.5 μL of forward primer (5 pmol L^-1), 0.5 μL of reverse primer (5 pmol L^-1), 1.0 μL of cDNA template (100 ng μL^-1), 14.8 μL of ddH₂O. The product of each amplification was analyzed by electrophoresis on 1% agarose gel (5 V cm^-1), 1xTBE buffer (89 mM Tris, 89 mM boric acid, 2 mM Na₂EDTA), using ethidium bromide staining (1 μg mL^-1).

For SSCP analysis, 2 μL PCR products were mixed with 5 μL denaturation solution (95% formamide, 0.5 mM EDTA, 0.025% xylene-cyanole and 0.025% bromophenol blue), heated for 10 min at 98°C and chilled on ice. Denatured DNA was subjected to PAGE (10%) (200x125x 1.00 mm) in 1xTBE buffer and constant voltage (110 V) for 12 h. The gel was stained with 0.1% silver nitrate. The PCR products from different SSCP genotypes were sub-cloned to T-vector (Promega) and sequenced by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd, Shanghai, China. For the same genotype, 3 samples from different individuals were sequenced independently at least.

Statistical analysis: All data were analyzed by ANOVA procedure of the statistical software SPSS version 17.0. The association between GH gene and productive traits of three goose breeds were analyzed by linear model and calculated using the least square method.

Table 1:	Primers used in PCR amplification

A fixed model was adopted according to the factors that affect phenotypic traits by using the following equation:

Where:

RESULTS AND DISCUSSION

The PCR amplification of goose GH gene encoding region: About 5 primers were designed to amplify the 5 exon sequence. The results showed there was specific and clear band for each exon, respectively, the PCR product could be used for PCR-SSC P analysis.

Detection of the goose GH genetic polymorphism SSCP detection of GH gene exon: PCR-SSCP results showed the genetic polymorphism was detected only at exon 2 and 4. For exon 2 there were 4 alleles (named A, B, C and D) and 10 genotypes were observed (Fig. 1), defined AA, BB,CC, DD, AB, AC, AD, BC, BD and CD genotype, respectively. For exon 4 there were 2 alleles (named A and B) and 3 genotypes were detected (Fig. 2), defined AA, BB and AB genotype, respectively.

Fig. 1:	SSCP analysis of PCR products of the exon 2 of GH gene (11: AA; 2: BB; 12: CC; 5: DD; 3: AB; 4: AC; 7: AD; 8: BC; 9: BD; 1, 6, 10: CD)

Fig. 2:	SSCP analysis of PCR products of the exon 4 of GH gene (1: AA; 4: BB; 2, 3, 5: AB)

Fig. 3:	Polymorphisms in the exon 2 of GH gene

Sequencing of different GH exon homozygous genotype: DNA sequencing analysis showed the sequence of AA genotype at exon 2 was the same to the sequence (AB158760) in Gene bank, two novel SNP (Fig. 3) was found in the BB genotype.

According to the mutation nomenclature (www.genomic.unimelb.edu.au/mdi/mutnomen or www. hgvs.org/mutnomen) and its extensions and suggestions (Den Dunnen and Antonarakis, 2000), the mutations were described as c.39 T>C and c.74 T>C which would result in synonymous mutation at 39 bp however, there was just one mutation found at CC (c.39 T>C) and DD genotype (c.74 T>C), respectively which resulted in the missense mutation (Ala→Val) at 74 bp. For the exon 4, the sequence of AA genotype was the same to the sequence (AB158760) in Gene bank, two novel SNP was found in BB genotype, c.291 C>T and c.297C>G (Fig. 4) which all resulted in synonymous mutation.

Genotypes and allele frequency in different goose group: The genotype and alleles frequency was shown in Table 2. For the P2 locus, the dominant alleles was A and D for three goose breeds.

Fig. 4:	Polymorphisms in the exon 4 of GH gene

Table 2:	Genotypes and allele frequency distribution in different goose group
χ2 0.05 (2) = 5.99, χ2 0.01 (2) = 9.21;1:χ2 0.05 (9) = 16.92; (number in the bracket means number of genotypes)

Table 3:	χ2 test of genotypes distribution in different goosegroups
χ20.05 (2) = 5.99, χ20.01 (2) = 9.21 χ2 0.05 (9) = 16.92, χ20.01 (9) = 21.67

The χ²-test showed that the genotype distributions at P2 locus was in agreement with Hardy-Weinberg equilibrium (p>0.05). For the P4 locus, B allele showed the highest frequency at Shitou goose group then B allele was the dominant allele; A allele was the highest at Wan-xi white and Zi goose group so it was the dominant allele.

Table 4:	Effect of the source of variation on the value of body weight
p<0.05, **p<0.01

Table 5:	Comparison of body weight value in different groups
The same superscripts in the same line means not significant difference (p>0.05), different superscripts means significant difference (p<0.01)

The χ²-test showed that the genotype distributions at P4 locus was in agreement with Hardy-Weinberg equilibrium (p>0.05). P2 and P4 were used to amplify different exon region then leaded to the different genotype for three different goose population. It was clear to show from Table 3, the distributions of the P2 locus were significantly different (p<0.01) among the goose populations except for Zi goose and Wan-xi white goose (p>0.05). The distributions of the P4 locus were significantly different (p<0.01) except for Zi goose and Shitou goose (p>0.05).

Least square analysis of the genotype and the body weight: In this study, the association of the GH gene polymorphism with the body weight was shown (Table 4), the result showed that the group effect of the body weight was significantly different (p<0.01) in the two locus among the group, the genotype effect of that was significantly different (p<0.01) in P2 locus. The effect of genotype-group interaction was not significantly different (p>0.05) but P4 locus had no genotype effect (p>0.05). In addition, the body weight of different breeds was analyzed statistically using analysis of variance and compared by way of Duncan in this study (Table 5). The results indicated the body weight of every week of age was significantly different (p>0.05) in the three goose group and the general trend was Shitou goose> Wan-xi white goose> Zi goose.

That of Shitou goose was the highest and greater than Wan-xi white goose and Zi goose (p<0.01). The genotype effect of the body weight at P4 locus was shown in Table 6 using the least squares. In the three goose groups, the general trend of body weight was AA> BB> AB but the AA genotype was >BB genotype in Zi goose group.

Growth hormone gene polymorphism in White Leghorn, Bantamised WLH and Bantam birds selected for egg production and its correlation with age at first egg, body weights and egg production. There were significant (p<0.05) differences among the three breed groups for body weights, age at first egg and egg production (Kuhnlein et al., 1997). In this study, researchers assessed the interaction among GH gene polymorphism and different groups and body weight in Chinese local goose at 6-9 weeks of age.

Table 6:	Least square means standard and deviation of different genotypes in P4 locusfor body weight
The same superscripts in the same line means not significant difference (p>0.05), different superscripts means significant difference (p<0.05)

To the knowledge, the present findings genotypes and groups had an great effect on body weight but there was no interactive effect between genotypes and groups. The body weight in different group analysis showed that there were significant difference among three goose.

The average of Shitou goose body weight was the largest, the second one was Wanxi white goose body weight which was larger than Zi goose. Then Analysis of genotypes for exon 2 and 4 and its correlation with body weight showed that exon 4 presented significant difference on body weight at 6-9 weeks. There were no significant differences in the 6-9 weeks body weight among the different genotype at exon 2 locus. The genotypes at exon 2 locus did not affect body weight. The average of body weight for the AA genotype was significantly higher than those for the BB and AB genotypes in exon 4 locus.

Therefore, AA genotype groups was the best groups for the selection which can increase the growth rate. It can draw a conclusion from this study that GH could be a candidate gene for a quantitative trait locus in goose.

CONCLUSION

This study was conducted to study the polymorphism of GH gene in goose. There were two nucleotide mutations at P2 and P4 locus, respectively which resulted in ten genotypes and three genotypes. The χ² test showed that the genotype distributions of GH gene were in agreement with Hardy-Weinberg equilibrium in P2 and P4 locus (p>0.05). The least square on genotypes and groups analysis showed that there were significant differences (p<0.01) on growth traits in different groups however, only P4 locus have genotypes effect, the average value of AA genotype was the highest therefore, AA genotype could be the favorable marker for early breeding selection for high product trait of local goose.

ACKNOWLEDGEMENTS

This study was supported by Mordern Agricultural Industry Technology System Special Fund (nycytx-45-04), National Science and Technology Planning Project (2006BDA01A09 and 2006BDA14B06) and Provincial Universities Natural Science Basic Research Project (07KJB230138) in Jiangsu, China.