山羊生长分化因子9基因外显子2的PCR-SSCP分析

PCR-SSCP Analysis on Exon 2 of Growth Differentiation Factor 9 Gene in Goats

目的寻找与产羔数相关的遗传标记,为山羊高繁殖力的标记辅助选择提供科学依据。方法以控制Belclare绵羊和Cambridge绵羊高繁殖力的生长分化因子9(growthdifferentiationfactor9,GDF9)基因为候选基因,根据绵羊GDF9基因序列设计4对引物,采用PCR-SSCP技术检测GDF9基因外显子2在高繁殖力山羊品种(济宁青山羊)以及低繁殖力山羊品种(波尔山羊、文登奶山羊、辽宁绒山羊、北京本地山羊)中的单核苷酸多态性,同时研究该基因对济宁青山羊高繁殖力的影响。结果山羊与绵羊的GDF9基因外显子2核苷酸序列同源性为99%(955/965)。引物1和引物4存在多态性。对于引物1扩增片段,5个山羊品种均检测到AA、AB和BB3种基因型,测序分析发现外显子2第26bp处有1个G→A的单碱基突变,但没有引起氨基酸改变;济宁青山羊A等位基因频率为0.9128,B等位基因频率为0.0872;AA基因型济宁青山羊产羔数最小二乘均值比AB基因型的多0.54只(P<0.01),比BB基因型的多0.63只(P<0.01)。对于引物4扩增片段,5个山羊品种均检测到CC、CD和DD3种基因型,测序分析发现外显子2第792bp处有1个G→A的单碱基突变并且导致缬氨酸改变为异亮氨酸;济宁青山羊C等位基因频率为0.9266,D等位基因频率为0.0734;CC基因型济宁青山羊产羔数最小二乘均值比CD基因型的多0.57只(P<0.01),比DD基因型的多0.62只(P<0.01)。结论初步表明GDF9基因可能是控制济宁青山羊多胎性能的一个主效基因或是与之存在紧密遗传连锁的分子标记。

[ Objective ] The objectives of the present study were firstly to detect polymorphisms in the growth differentiation factor 9 （GDF9） gene, and secondly to investigate the association between GDF9 gene and high prolificacy in Jining Grey goats. These results will provide a scientific basis for marker-assisted selection for high prolificacy in goats. [Method] The GDF9 gene which controls the fecundity of Belclare and Cambridge ewes was studied as a candidate gene for the prolificacy in Jining Grey goats According to the sequence of ovine GDF9 gene, four primer pairs were designed to detect single nucleotide polymorphism of exon 2 of GDF9 gene in both high fecundity goat breed （Jining Grey goat） and low fecundity goat breeds （Boer goat, Wendeng dairy goat, Liaoning Cashmere goat, Beijing native goat） by PCR-SSCP. [Result] The results indicated that the homology of nucleotide sequence of exon 2 of GDF9 gene between goats and sheep was 99 percent. The products amplified by primer 1 and primer 4 displayed polymorphism. For primer 1, three genotypes （AA, AB and BB） were detected in all five goat breeds. The sequencing results indicated that there was one single nucleotide mutation （G^26→A^26） at exon 2 of GDF9 gene in goats, but this mutation did not cause amino acid change. Frequency of allele A was 0.9128, frequency of allele B was 0.0872 in Jining Grey goats. The does with genotype AA had 0.54 （P〈0.01） or 0.63 （P〈0.01） kids more than those with genotype AB or BB in Jining Grey goats, respectively. For primer 4, three genotypes （CC, CD and DD） were detected in all five goat breeds. The sequencing results showed that there was one single nucleotide mutation （G^792→A^792） at exon 2 of GDF9 gene in goats, and this mutation resulted in an amino acid change： valine→isoleucine. Frequency of allele C was 0.9266, frequency of allele D was 0.0734 in Jining Grey goats. The does with genotype CC had 0.57 （P〈0.01） or 0.62 （P〈0.01） kids more than those with genotype CD or DD in Jining Grey goats, respectively. [ Conclusion ] These results preliminarily showed that the GDF9 gene is either a major gene that influences the prolificacy in Jining Grey goats or a molecular marker in close linkage with such a gene.