Deceptive combined effects of short allele dominance and stuttering: an example withIxodes scapularis, the main vector of Lyme disease in the U.S.A.

Abstract

Null alleles, short allele dominance (SAD), and stuttering increase the perceived relative inbreeding of individuals and subpopulations as measured by Wright’s FIS and FST. Ascertainment bias, due to such amplifying problems are usually caused by inaccurate primer design (if developed from a different species or a distant population), poor DNA quality, low DNA concentration, or a combination of some or all these sources of inaccuracy. When combined, these issues can increase the correlation between polymorphism at concerned loci and, consequently, of linkage disequilibrium (LD) between those. In this note, we studied an original microsatellite data set generated by analyzing nine loci in Ixodes scapularis ticks from the eastern U.S.A. To detect null alleles and SAD we used correlation methods and variation measures. To detect stuttering, we evaluated heterozygote deficit between alleles displaying a single repeat difference. We demonstrated that an important proportion of loci affected by amplification problems (one with null alleles, two with SAD and three with stuttering) lead to highly significant heterozygote deficits (FIS=0.1, p-value<0.0001). This occurred together with an important proportion (22%) of pairs of loci in significant LD, two of which were still significant after a false discovery rate (FDR) correction, and some variation in the measurement of population subdivision across loci (Wright’s FST). This suggested a strong Wahlund effect and/or selection at several loci. By finding small peaks corresponding to previously disregarded larger alleles in some homozygous profiles for loci with SAD and by pooling alleles close in size for loci with stuttering, we generated an amended dataset. Except for one locus with null alleles and another still displaying a modest SAD, the analyses of the corrected dataset revealed a significant excess of heterozygotes (FIS=-0.07 as expected in dioecious and strongly subdivided populations, with a more reasonable proportion (19%) of pairs of loci characterized by significant LD, none of which stayed significant after the FDR procedure. Strong subdivision was also confirmed by the standardized FST’ corrected for null alleles (FST’=0.19) and small effective subpopulation sizes (Ne=7).

Altitude and hillside orientation shapes the population structure of the Leishmania infantum vector Phlebotomus ariasi

“…Using regression approach we detected a marginally not significant Short Allele Dominance (SAD) for the Aria14 locus (p value = 0.0507). As SAD results from a preferential amplification of the shortest allele in heterozygous individuals 28 , the Aria14 microsatellite profile of each homozygous individual was checked again following recommendation suggested in De Meeûs et al 29 , corrected, and then analyzed using GENEMAPPER 4.0. After correction, SAD could not be detected on this locus any longer (p value = 0.0731).…”

“…Nevertheless, it was marginally not significant for Aria10 (p value = 0.0503) and highly significant for Aria15 (p value = 9.728e−06). Therefore, alleles close in size were pooled, avoiding pooling together only rare alleles (i.e., presence of at least one frequent allele in the pool) for these two loci as recommended 29 . As the Aria10 locus became monomorphic after pooling, this locus was removed from the analysis.…”

“…As the clustering method accuracy can vary depending on the statistical properties of specific software programs and datasets 29,42,63 , three different Bayesian clustering methods were used. First, a discriminant analysis of principal components (DAPC) 64 was performed using the adegenet 65 package for R 66 .…”

“…In the case of SAD, a negative correlation is expected between allele size and F IT 42 . In the case of significant stutter artifacts or SAD, the incriminated loci were corrected using the method described by De Meeûs et al 29 . Stuttering was addressed by pooling alleles close in size.…”

Developing and quality testing of microsatellite loci for four species of Glossina

“…These markers are frequent in the genome of most eukaryotes, easy to handle and highly polymorphic (Katti et al, 2001), except in some flying vertebrates like birds and bats where weight loss evolution explains their rarity (Primmer et al, 1997). Moreover, their extensive use in various organisms over a fairly long period of time have allowed the development of efficient methods to detect and cure allele mis-scoring due to the more or less frequent amplification problems met, such as null alleles, short allele dominance (SAD), allelic dropout, and stuttering (Chapuis and Estoup, 2007;Wang et al, 2012;Séré et al, 2014;Séré et al, 2017;De Meeûs, 2018;De Meeûs et al, 2019a;Manangwa et al, 2019). More modern and numerous single nucleotide polymorphisms (SNPs) may be used instead, but one can see many reasons why SNPs may be less suitable than microsatellites in population genetics studies.…”