奶牛(Bos taurus)的繁殖性能与机体能量代谢密切相关,过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor γ,PPARγ)是由PPARG基因编码的配体激活的核转录因子,在调控动物能量稳态、繁殖生理和免疫应答等过程中发挥关键性作用,PPARG是奶牛繁殖性状的重要候选基因.本实验采用聚合酶链式反应-单链构象多态(polymerase chain reaction products-single strand conformation polymorphism,PCR-SSCP)技术并结合测序,检测中国荷斯坦牛PPARG基因exon7区Q448H错义突变,并分析其对中国荷斯坦牛繁殖性状的影响.结果表明,PPARG基因exon7共存在2个等位基因3种基因型,等位基因G/T在低代杂种、高代杂种和纯种荷斯坦牛群体中的频率分别为0.65/0.35、0.75/0.25和0.69/0.31,优势基因型为GG型;3个实验群体在该位点均处于中度多态.测序结果显示,与普通牛PPARG基因序列(NC_007320.6)相比,T等位基因在64 947位核苷酸处发生了G→T的碱基突变,导致第448位氨基酸由谷氨酰胺变为组氨酸,而G等位基因与NC_007320.6序列相同.最小二乘法分析表明,GG型产后第一次配种天数(days to first breeding,DFB)显著低于GT型(P＜0.05)和TT型(P＜0.01),标记对产后第一次配种天数的表型贡献率为3.82％;GG型空怀天数(days open,DO)显著低于GT型(P＜0.05),标记对空怀天数的表型贡献率为2.55％;GG型第1胎次配妊次数(number of services per conception,NSC)显著低于GT型(P＜0.01),标记对第1胎次配妊次数的表型贡献率为2.24％;第2胎次NSC显著低于GT型和TT型(P＜0.05),标记对第2胎次配妊次数的表型贡献率为3.46％;各基因型之间初产月龄(age at first calving,AFC)差异不显著(P＞0.05).对于G等位基因,DFB、DO和NSC(第2胎次)的加性效应分别为-6.97 d、-11.2 d和-0.215次.基因替代效应分别为-8.83 d、-15.70 d和-0.25次,即每个G等位基因替代T等位基因会导致产后第一次配种天数提前8.83 d,空怀天数缩短15.7 d,配妊次数降低0.25次,等位基因G为高繁殖性能优势基因.PPARG基因Q448H突变位点可作为中国荷斯坦牛繁殖性状的分子标记.本研究为中国荷斯坦牛繁殖性状的标记辅助选择提供了理论依据.%Reproductive performance in dairy cows is highly correlated with energy metabolism.The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated nuclear transcription factor that plays an important role in regulating the processes of energy homeostasis,reproductive,and immune physiology.A potentially important candidate gene for reproductive traits of dairy cows is PPARγ,which is encoded by the PPARG.In the present study,a Q448H mutation of the PPARG gene exon7 in Chinese Holstein cattle was detected with polymerase chain reaction products-single strand conformational polymorphism (PCR-SSCP) and DNA sequencing,and assessed through reproductive traits.Results demonstrate the presence of two alleles and 3 genotypes in the locus of the PPARG gene exon7.The 2 alleles were named G and T.The allelic frequencies of the G/T alleles in the three groups were 0.65/0.35,0.75/0.25,and 0.69/0.31 for the Low-Hybrids,Improved-Hybrids,and Pure Holstein populations,respectively;the GG genotype was preponderant.Except for the Improved-Hybrids (P＜0.01),Low-Hybrids and Pure Holsteins were in Hardy-Weinberg equilibrium (P＞ 0.05) at this locus.Polymorphism information content was moderately high for Low-Hybrids,Improved-Hybrids,and Pure Holsteins at 0.35,0.31,and 0.34,respectively.Comparing this data with database sequence of Bos taurus (Accession #:NC_007320.6),the mutation of G to T occurred at the 64 947 bp of allele T,which resulted in the substitution of glutamine into histidine,allele G was the same sequence as that of NC_007320.6.Analysis by least squares methodology showed that days-to-first-breeding (DFB) with genotype GG were significantly lower than that with the genotype GT (P＜0.05) and TT (P＜0.01).The days open (DO) with the genotype GG were significantly lower than that with genotype GT (P＜0.05).The number of services-per-conception (NSC),with genotype GG were significantly lower than that with the genotype GT (P＜0.01) at first lactation,and with the genotypes GT (P＜0.05) and TT (P＜0.05) at second lactation.It can be explained by the PPARG gene Q448H mutation that phenotypic variances were 3.82％,2.55％,2.24％,and 3.46％ for the DFB,DO,and NSC at first and second lactations,respectively.There were no differences in the ages-at-first-calving (AFC) between any genotypes (P＞0.05).Additive effects of the G allele were-6.97 d,-11.2 d,and-0.215 t for DFB,DO,and NSC at second lactation,respectively.DFB and DO were reduced by 8.8 days and 15.7 days,respectively,while NSC was reduced by a factor of 0.25 with the allelic substitution of T to G,thereby indicating that the G allele of PPARG gene could benefit from further investigation as its role appears to be associated with reproductive performance.We conclude that the PPARG gene Q448H mutation could be used as a genetic marker for studies seeking to improve reproductive traits in Chinese Holstein cattle.This study has provided the theoretical foundation for maker-assistant-selection (MAS) of reproductive performance in Chinese Holstein cattle.
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