The Effect of microsatellite number and motif type on estimation of population parameters in genetic diversity studies i

Microsatellites are repetitive regions in DNA including homogeneous array of mono, di, tri, tetra, penta and hexa nucleotides with length of less than 1 Kbp which are non-randomly distributed in genome. The number and density of microsatellites is vary within species even in very close spices such as humans and chimpanzees. The frequency of microsatellite motifs and their mutation rate is reported differently in various organisms. In mammalian genomes di-nucleotide microsatellites are the more abundant type following by mono and tetra microsatellite motifs as next. Tri microsatellites are more frequent in plants. However, the effect of variety in microsatellite motifs on genetic diversity or population structural parameters is a topic that has received less attention.

In the present study with using the 36 VCf file of microsatellite markers extracted from whole genome of Iranian sheep “Ovis aries and Ovis orientalis” by NGS, the total number of 163973 microsatellite markers were detected. The distribution of Ovis aries samples is from north-west part of Iran and ovis orientalis samples are belongs to central part and north-west of Iran. After rearrangement and filtration on data file using Samtools and VCFtools softwares, we classified the whole markers on four different motif types including di- tri- and tetra-nucleotide microsatellites and a file includes all three microsatellite types. Several subsets including 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400 and 500 markers were generated from each microsatellite motif types using a R script. Six common genetic diversity parameters including observed and expected heterozygosity, Nei diversity index, Shanon index, Allelic richness and FIS were calculated for each different subset of number and motif type of microsatellites in MSA (V.4.05) software. 10 replications were considered for each parameter. The mean and variance were calculated among 10 replications and results were represented by boxplots using R (v.3.3.3). The statistical investigation of parameter estimation differences using different microsatellite number and motif types were analyzed using ANOVA for testing the hypothesis of equality of means in R (v.3.3.3).

Estimation of all six parameters revealed various results using different number of loci as well as motif types. Additionally, the results revealed higher values for parameters estimated with di microsatellite motifs compared to others. In addition, the highest and lowest value for most parameters were obtained by 40-di and 10-tri/tetra microsatellites respectively. The statistical significance on findings of parameter values using different number/motif of microsatellite markers were analyzed using ANOVA in R (v.3.3.3). In the case with significant results, Tukey Honestly Significant Difference test was used to test pairwise contrasts between different subsets.

Our result proposes a better application of di microsatellites for genetic diversity studies in sheep populations. Moreover, results showed that for stable estimation of population parameters in genetic diversity studies a minimum of 50 microsatellite loci are needed.