Sampling for Microsatellite-Based Population Genetic Studies: 25 to 30 Individuals per Population Is Enough to Accurately Estimate Allele Frequencies

Abstract

One of the most common questions asked before starting a new population genetic study using microsatellite allele frequencies is “how many individuals do I need to sample from each population?” This question has previously been answered by addressing how many individuals are needed to detect all of the alleles present in a population (i.e. rarefaction based analyses). However, we argue that obtaining accurate allele frequencies and accurate estimates of diversity are much more important than detecting all of the alleles, given that very rare alleles (i.e. new mutations) are not very informative for assessing genetic diversity within a population or genetic structure among populations. Here we present a comparison of allele frequencies, expected heterozygosities and genetic distances between real and simulated populations by randomly subsampling 5–100 individuals from four empirical microsatellite genotype datasets (Formica lugubris, Sciurus vulgaris, Thalassarche melanophris, and Himantopus novaezelandia) to create 100 replicate datasets at each sample size. Despite differences in taxon (two birds, one mammal, one insect), population size, number of loci and polymorphism across loci, the degree of differences between simulated and empirical dataset allele frequencies, expected heterozygosities and pairwise FST values were almost identical among the four datasets at each sample size. Variability in allele frequency and expected heterozygosity among replicates decreased with increasing sample size, but these decreases were minimal above sample sizes of 25 to 30. Therefore, there appears to be little benefit in sampling more than 25 to 30 individuals per population for population genetic studies based on microsatellite allele frequencies.

Prelude to a panzootic: Gene flow and immunogenetic variation in northern little brown myotis vulnerable to bat white-nose syndrome

“…Some analyses of genetic diversity require individuals to be assigned to prior groups (rarified allelic richness and expected heterozygosity), and some can provide greater resolution when given prior information on likely group membership (i.e., STRUCTURE, Pritchard et al 2000). For these analyses, we clustered samples collected at nearby sampling locations into sampling areas, aiming to (1) maximize the number of distinct sampling areas, and (2) ensure that each defined group of samples contained >25 individuals where possible, to avoid biasing frequency-based analyses with inadequate sample sizes (Hale et al 2012). In total, we defined 23 sampling areas from 13 ecozones for analysis based on geographic clustering and on physiographic variations across our study area (Table 1; Fig.…”

Restriction to gene flow is associated with changes in the molecular basis of pyrethroid resistance in the malaria vector Anopheles funestus

“…The 17 microsatellite markers spanning the entire genome were genotyped consistently in all populations, demonstrating robustness of the multiplex design. Each population analyzed contained ≥36 individuals (≥72 alleles), which is sufficient for accurately estimating allele frequencies (9). The loci FUNR, FUNQ, and AFND12 showed an excess of one allele.…”

A note on the distribution of genetic diversity of Anglo-Nubian goats in central-northern farms of Piauí, Brazil

“…Some studies using microsatellite markers sampled more than 50 individuals from each population (Li et al, 2002), more than used in the presented study. However, according to Hale et al (2012), for precise estimates of allele frequencies and genetic diversity, no more than 25 to 30 individuals are needed per population. Thus, the possibility that the breed has a narrow genetic base in the studied region cannot be ruled out, due to indiscriminate crossings without technical information.…”