雪峰乌骨鸡HSP70基因多态性与精液品质关联分析

旨在研究雪峰乌骨鸡HSP70基因多态性及其与鸡冻精品质的关系,以期筛选出鸡精液抗冻性相关分子标记,为鸡精液冷冻保存及其分子育种提供技术支撑。选取100只同一批次、性成熟的白羽雪峰乌骨种公鸡,测定采精量、精子密度和冻精活力等指标。翅静脉采血,提取基因组DNA并扩增HSP70基因,构建精液品质低、中、高3个混池并测序。单个样本测序后,比对测序峰图,完成基因分型,统计不同基因型个体数,并进一步进行精液品质关联分析。结果发现:HSP70基因436 bp处存在G>A突变,G为优势基因,将群体分成AA、AG和GG这3种基因型,各基因型频率依次为22%、44%和34%,该位点遗传多样性分析PIC=0.371 4,为中度多态位点,Hardy-Weinberg平衡适合性检验得χ²=1.148 0(P>0.05),该群体符合Hardy-Weinberg平衡规律。基因多态与精液品质关联分析表明,GG基因型个体采精量和精子密度极显著(P<0.01)高于AG型、AA型个体,且冻后活力显著(P<0.05)高于AG型个体,极显著(P<0.01)高于AA型个体;AG型个体采精量和精子密度极显著(P<0.01)高于AA型个体,冻精活力差异不显著(P>0.05)。综上所述,HSP70基因突变G/A位点与鸡冻精品质显著相关,可以作为雪峰乌骨鸡个体精液抗冻性的候选基因,基因型GG的个体精液更具抗冻性,其次为AG型个体。

Correlation analysis of HSP70 gene polymorphism and semen quality in Xuefeng Black-bone chicken

To study HSP70 gene polymorphism of Xuefeng black-bone chicken and its relationship with frozen semen quality of chicken. Molecular markers related to freezing resistance of chicken semen were screened out to provide technical support for freezing preservation and molecular breeding of chicken semen. One hundred mature white Xuefeng black bone roosters of the same batch were selected. The semen volume, semen density and frozen semen vitality of rooster were measured. Blood was collected from the pterygium vein. gDNA was extracted from the blood sample and HSP70 gene was amplified. Three mixed pools of low, medium and high semen quality were constructed and sequenced. After sequencing a single sample, the genotyping was completed by comparing the sequencing peak diagram. The number of individuals with different genotypes was counted and the correlation analysis of semen quality was further carried out. The results showed that there was a mutation G>A at 436 bp in HSP70 gene, and G was the dominant gene. The population was divided into AA, AG and GG genotypes with the frequencies of 22%, 44% and 34%, respectively. The genetic diversity analysis PIC of this locus was 0.371 4, which was a moderate polymorphic locus. Hardy-Weinberg equilibrium fitness test showed χ²=1.1480 (P>0.05), which showed that the population conformed to Hardy-Weinberg equilibrium law. Correlation analysis between gene polymorphism and semen quality showed that the sperm volume and semen density of GG genotype individuals were significantly higher than those of AG type and AA type individuals (P<0.01). The viability after freezing of GG genotype individuals was significantly higher than that of AG type individuals (P<0.05), and was significantly higher than that of AA type individuals (P<0.01). The semen collection volume and semen density of AG type individuals were significantly higher than those of AA type individuals (P<0.01), and the difference of frozen semen activity was not significant. To sum up, the mutant G>A site of HSP70 gene was significantly related to the frozen semen quality of chicken. The locus could be used as a candidate gene for individual semen antifreeze of Xuefeng black-bone chicken. The individual with genotype GG had the best semen anti-freezing, followed by AG type individual.