Microsatellites for the Marsh Fritillary Butterfly: De Novo Transcriptome Sequencing, and a Comparison with Amplified Fragment Length Polymorphism (AFLP) Markers

Abstract

BackgroundUntil recently the isolation of microsatellite markers from Lepidoptera has proved troublesome, expensive and time-consuming. Following on from a previous study of Edith's checkerspot butterfly, Euphydryas editha, we developed novel microsatellite markers for the vulnerable marsh fritillary butterfly, E. aurinia. Our goal was to optimize the process in order to reduce both time and cost relative to prevailing techniques. This was accomplished by using a combination of previously developed techniques: in silico mining of a de novo assembled transcriptome sequence, and genotyping the microsatellites found there using an economic method of fluorescently labelling primers.Principal FindingsIn total, we screened nine polymorphic microsatellite markers, two of which were previously published, and seven that were isolated de novo. These markers were able to amplify across geographically isolated populations throughout Continental Europe and the UK. Significant deviations from Hardy-Weinberg equilibrium were evident in some populations, most likely due to the presence of null alleles. However, we used an Fst outlier approach to show that these markers are likely selectively neutral. Furthermore, using a set of 128 individuals from 11 populations, we demonstrate consistency in population differentiation estimates with previously developed amplified fragment length polymorphism (AFLP) markers (r = 0.68, p<0.001).SignificanceRapid development of microsatellite markers for difficult taxa such as Lepidoptera, and concordant results with other putatively neutral molecular markers, demonstrate the potential of de novo transcriptional sequencing for future studies of population structure and gene flow that are desperately needed for declining species across fragmented landscapes.

Host‐associated genomic differentiation in congeneric butterflies: now you see it, now you do not

“…; Smee et al . ). These two studies developed 9 loci for E. aurinia (3.4 ± 1.2 alleles/locus) and 10 loci for E. editha (5.3 ± 2.3 alleles/locus) and used the same individuals that were previously genotyped for AFLPs, but also added additional samples gathered between 2008 and 2010.…”

“…; Smee et al . ), we used averages of the θ st and F st matrices where both values were available. Because AFLP data were from an earlier and less comprehensive survey, they were missing for several populations, forcing us to rely on microsatellite data only in those cases.…”

“…That analysis used four primer combinations for E. aurinia and three primer combinations for E. editha, producing 318 polymorphic loci in E. aurinia and 547 polymorphic loci in E. editha. The other data sets were from EST-derived microsatellites identified in two previous studies, which developed the molecular markers, and genotyped the populations used in the present study, but did not conduct analysis of HAD (Mikheyev et al 2010;Smee et al 2013). These two studies developed 9 loci for E. aurinia (3.4 AE 1.2 alleles/locus) and 10 loci for E. editha (5.3 AE 2.3 alleles/locus) and used the same individuals that were previously genotyped for AFLPs, but also added additional samples gathered between 2008 and 2010.…”

“…h st is an estimator of F st based upon the proportion of allele frequencies shared between individuals in different subpopulations (Weir & Cockerham 1984). As these two statistics are analogous and strongly correlated in our data (Mikheyev et al 2010;Smee et al 2013), we used averages of the h st and F st matrices where both values were available. Because AFLP data were from an earlier and less comprehensive survey, they were missing for several populations, forcing us to rely on microsatellite data only in those cases.…”

Efficient development of highly polymorphic microsatellite markers based on polymorphic repeats in transcriptome sequences of multiple individuals

“…Additionally, being multi-allelic, they are powerful for parentage analysis and haplotyping, particularly for mapping in polyploids as they allow detecting multiple alleles at the same locus on all homologous chromosomes . Despite the advent of SNP markers, recent studies in various plant and animal genera, for instance, Cucurbita (Barzegar et al 2013), Euphydryas (Smee et al 2013), Lilium (Yuan et al 2013), Medicago (Zitouna et al 2013), Pinus (Iwaiz-umi et al 2013), Portunus (Guo et al 2013), Scatophagus , Triticum (Ansari et al 2013) and Vitis (Doulati-Baneh et al 2013) indicate that microsatellite markers are still extensively being developed as a molecular tool for various purposes.…”