济宁青山羊GDF9基因外显子2多态性检测

Polymorphism Analysis on Exon 2 of Growth Differentiation Factor 9 Gene in Jining Grey Goat

对山羊生长分化因子9(growth differentiation factor 9,GDF9)基因进行单核苷酸多态性检测,为进一步研究GDF9基因与山羊产羔数的相关性奠定理论基础。根据GDF9基因序列(GenBank:EF 446168),设计3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)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。引物1和引物3存在多态性。对于引物1扩增片段,3个山羊品种均检测到AA、AB和BB 3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0.8917,B等位基因频率为0.1083;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0.539只(P<0.01),比BB基因型的多0.629只(P<0.01)。对于引物3扩增片段,3个山羊品种均检测到CC、CD和DD 3种基因型,测序分析发现外显子2第795bp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0.9167,D等位基因频率为0.0833;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0.568只(P<0.01),比DD基因型的多0.62只(P<0.01)。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。

The single nucleotide polymorphisms of GDF9 （growth differentiation factor 9） gene on goats was tested,which provided theoretical basis for studying the correlations among GDF9 gene with lambing number.According to the sequence of GDF9gene （GenBank：EF 446168）,three primer pairs were designed to detect single nucleotide polymorphism of exon 2 ofGDF9 gene in both high fecundity goat breed （Jining Grey goat） and low fecundity goat breeds （Laoshan dairy goat,Laiwu native goat） by PCR-SSCP.The products amplified by primer 1 and primer 3 displayed polymorphism.For primer 1,three genotypes （AA,AB and BB） were detected in all three goat breeds.The sequencing results indicated that there was one single nucleotide mutation （G26→A26） at exon 2 of GDF9 gene in goats,but this mutation did not cause amino acid change.Frequency of allele A was 0.8917,frequency of allele B was 0.1083 in Jining Grey goats.The does with genotype AA had 0.539 （P＜0.01） or 0.629 （P＜0.01） kids more than those with genotype AB or BB in Jining Grey goats,respectively.For primer r3,three genotypes （CC,CD and DD） were detected in all fivegoat breeds.The sequencing results showed that there was one single nucleotide mutation （G795→A795） at exon 2 of GDF9 gene in goats,and this mutation resulted in an amino acid change：valine→isoleucine.Frequency of allele C was 0.9167,frequency of allele D was 0.0833 in Jining Grey goats.The does with genotype CC had 0.568 （P＜0.01） or 0.62 （P＜0.01） kids more than those with genotype CD or DD in Jining Grey goats,respectively.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.