[Impact of the number of microsatellite markers on the analysis of population genetic diversity of ].

Objective: To examine the impact of different numbers of microsatellite markers on the analysis of population genetic diversity of , so as to provide insights into studies on the population genetic diversity of .

Methods: snails were collected from a wasteland in Gong'an County, Hubei Province, and 37 -infected snails were identified using the cercarial shedding method. A single cercaria released from each -infected 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 cercariae DNA was analyzed with observed number of alleles (), effective number of alleles (), observed heterozygosity (), expected heterozygosity (), 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 , the number of microsatellite markers was sequentially assigned from 1 to 24, and the mean and standard deviation of were calculated for populations at different locus numbers. In addition, the coefficient of variation () of allelic number (defined as the ratio of the standard deviation to the mean) was determined, and the variation in with increasing microsatellite locus numbers was analyzed.

Results: Genomic DNA from 345 cercariae was selected for genotyping of 24 microsatellite markers, and all 24 microsatellite loci met linkage equilibrium (standardized linkage disequilibrium coefficient ' < 0.7, < 0.3) and deviated from Hardy-Weinberg equilibrium ( < 0.001). The mean , , and were 27.46 ± 2.18, 12.46 ± 0.95, 0.46 ± 0.03, and 0.91 ± 0.01 for 24 microsatellite loci in 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 - 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 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 remarkably reduced. In addition, the standard deviation of at 18 loci was less than 5% of the mean at 24 loci, with a of 4.6%.

Conclusions: The number of microsatellite loci significantly affects the population genetic diversity analysis of . Eighteen or more microsatellite loci are recommended for analysis of the population genetic diversity of under the current conditions of low-prevalence infection and unbalanced genetic distribution of .