Abstract: Objective　To examine the impact of different numbers of microsatellite markers on the analysis of population genetic diversity of Schistosoma japonicum, so as to provide insights into studies on the population genetic diversity of S. japonicum. Methods　Oncomelania hupensis snails were collected from a wasteland in Gong'an County, Hubei Province, and 37 S. japonicum⁃infected O. hupensis snails were identified using the cercarial shedding method. A single cercaria released from each S. japonicum⁃infected O. hupensis snail was collected, and 10 cercariae were randomly collected from DNA extraction. Nine previously validated microsatellite loci and 15 additional microsatellite loci screened from literature review and the GenBank database and confirmed with stable amplification efficiency were selected as molecular markers. Genomic DNA from cercariae was subjected to three multiplex PCR amplifications of microsatellite markers with the Type⁃it Microsatellite PCR kit, and genotyped using capillary electrophoresis. The population genetic diversity of S. japonicum cercariae DNA was analyzed with observed number of alleles (Na), effective number of alleles (Ae), observed heterozygosity (Ho), expected heterozygosity (He), and polymorphism information content (PIC), and tested for Hardy⁃Weinberg equilibrium (HWE) and linkage disequilibrium (LD). To further investigate the impact of the number of microsatellite loci on the population genetic diversity of S. japonicum, the number of microsatellite markers was sequentially assigned from 1 to 24, and the mean and standard deviation of Na were calculated for S. japonicum populations at different locus numbers. In addition, the coefficient of variation (CV) of allelic number (defined as the ratio of the standard deviation to the mean) was determined, and the variation in Na with increasing microsatellite locus numbers was analyzed. Results　Genomic DNA from 345 S. japonicum cercariae was selected for genotyping of 24 microsatellite markers, and all 24 microsatellite loci met linkage equilibrium (standardized linkage disequilibrium coefficient D′ < 0.7, r2 < 0.3) and deviated from Hardy⁃Weinberg equilibrium (P < 0.001). The mean Na, Ae, Ho and He were 27.46 ± 2.18, 12.46 ± 0.95, 0.46 ± 0.03, and 0.91 ± 0.01 for 24 microsatellite loci in S. japonicum cercarial populations, respectively, and PIC ranged from 0.85 to 0.96, indicating high genome⁃wide representativeness of 24 microsatellite loci. The mean value of Na-Ae was higher in genotyping with 9 previously validated microsatellite loci (19.88 ± 8.43) than with all 24 loci (14.99 ± 8.09). As the number of microsatellite loci increased, the mean Na showed no significant variation; however, the standard deviation gradually decreased. Notably, if the locus number reached 18 or more, the variation in the standard deviation of Na remarkably reduced. In addition, the standard deviation of Na at 18 loci was less than 5% of the mean Na at 24 loci, with a CV of 4.6%. Conclusions　The number of microsatellite loci significantly affects the population genetic diversity analysis of S. japonicum. Eighteen or more microsatellite loci are recommended for analysis of the population genetic diversity of S. japonicum under the current conditions of low⁃prevalence infection and unbalanced genetic distribution of S. japonicum.

Key words: Schistosoma japonicum, Cercaria, Population structure, Genetic diversity, Microsatellite locus, Allele, Genetic polymorphism

摘要： 目的　探究采用不同数量微卫星位点标记对日本血吸虫种群遗传多样性分析的影响，为日本血吸虫种群遗传学研究提供参考。方法　自湖北省公安县某地野外荒滩采集湖北钉螺，采用直管逸蚴法筛选出37只血吸虫感染性钉螺。分别收集每只感染性钉螺逸出的单条尾蚴，各随机挑选10条尾蚴提取DNA。以前期经大规模样本验证过的9个微卫星位点以及自参考文献和GenBank数据库中筛选且可稳定扩增的15个微卫星位点作为分子标记，利用Type⁃it 微卫星PCR试剂盒对上述尾蚴DNA进行3组8重微卫星PCR扩增，通过毛细管电泳检测样本基因型。对上述日本血吸虫尾蚴DNA进行种群遗传多样性分析，评估等位基因数（observed number of alleles，Na）、有效等位基因数（effective number of alleles，Ae）、观察杂合度（observed heterozygosity，Ho）、期望杂合度（expected heterozygosity，He）和多态信息含量（polymorphism information content，PIC）等多态性指标，采用Hardy⁃Weinberg平衡检验和连锁不平衡评估进行尾蚴种群遗传结构分析。此外，为进一步探究微卫星位点数量对日本血吸虫种群遗传多样性的影响，依次将微卫星位点数量设定为1～24个，计算不同位点数量时日本血吸虫种群Na均数及其标准差，并计算等位基因数变异系数，观察Na随微卫星位点数量增加的变化。结果　共选取345条日本血吸虫尾蚴DNA，用上述24个微卫星位点进行检测，结果显示全部位点均满足连锁平衡标准化［连锁不平衡系数（D′） < 0.7，r2 < 0.3）］，均偏离Hardy⁃Weinberg平衡（P < 0.001）。日本血吸虫尾蚴种群在24个微卫星位点的Na、Ae、Ho和He均值分别为27.46 ± 2.18、12.46 ± 0.95、0.46 ± 0.03和0.91 ± 0.01，PIC值为0.85～0.96，提示24个位点在全基因组微卫星水平上均具有较好的代表性。采用经前期验证的9个微卫星位点进行分析时，日本血吸虫种群Na-Ae均值为19.88 ± 8.43，高于使用全部24个位点分析时的结果（14.99 ± 8.09）。随着微卫星位点数量的增加，Na均值虽无明显变化，但标准差逐渐变小；尤其是当位点数为18个及以上时，标准差变化幅度明显减小；当位点数为18个时，Na标准差小于位点数为24个时Na均值的5%，变异系数为4.6%。结论　微卫星位点数量可显著影响日本血吸虫种群遗传多样性分析结果。在目前低感染率和血吸虫遗传分布不平衡的背景下，推荐选取≥ 18个微卫星位点进行日本血吸虫种群遗传多样性分析。

关键词: 日本血吸虫, 尾蚴, 种群结构, 遗传多样性, 微卫星位点, 等位基因, 基因多态性