Development of novel microsatellite markers for a specialist species of Lepidoptera, Boloria aquilonaris (Nymphalidae), based on 454 sequences

Abstract

Abstract. Microsatellites are the most common markers used in population and conservation genetic studies. However, their isolation is laborious and expensive. In some taxa, such as Lepidoptera, it is particularly difficult to isolate microsatellite markers due to the high similarity of the flanking regions of different loci and the presence of null alleles. Here we isolated microsatellites of the endangered butterfly Boloria aquilonaris using 454 GS-FLX Titanium pyro-sequences of biotin enriched DNA libraries and tested the success of cross-amplification on the sister-species B. eunomia. Fifteen polymorphic microsatellite loci were isolated in B. aquilonaris using initially 101 stringently designed primer pairs. Unlike in many other studies of microsatellite isolation in Lepidoptera, few null alleles were detected and only at very low frequencies. Additionally, the raw data set can still be used for the isolation of other microsatellite loci. None of the selected polymorphic loci for B. aquilonaris gave clear banding patterns for B. eunomia, although about 15 other loci gave promising banding patterns for the latter species. Low intra-and inter-specific transferability of developed markers in this study also lends support to the hypothesis that the evolution of the genome of Lepidoptera is dissimilar from that of other organisms. 129* Corresponding and current address:

Conservation genetics of a threatened butterfly: comparison of allozymes, RAPDs and microsatellites

“…allele frequencies were calculated from null homozygote frequencies assuming panmixia and corrected for dominance according to Lynch & Milligan [ 31 ] using TFPGA 1.3 ( http://www.marksgeneticsoftware.net/tfpga.htm ). Fifteen microsatellite loci were amplified in three multiplex reactions [ 24 ]. Multiplex amplifications were conducted using QIAGEN Multiplex PCR Kit (QIAGEN) on Veriti Thermal Cycler (Applied Biosystems) with the following protocol: 95°C for 15 min, followed by 32 cycles (94°C for 30 s, 56°C for 90 s, 72°C for 90 s), and 72°C for 30 min, terminating at 25°C.…”

“…However, next-generation sequencing offers the opportunity for more efficient microsatellite isolation than was previously possible [ 22 , 23 ]. The combination of a biotin-enrichment protocol and high-throughput pyrosequencing has recently resulted in the successful isolation of microsatellites for several butterfly species, including the cranberry fritillary Boloria aquilonaris [ 24 ], a butterfly species of major conservation concern.…”

“…Following the successful isolation of 15 microsatellites loci for B. aquilonaris [ 24 ], here we analyze the genetic diversity and structure of eight populations located in four different landscapes. Analyses are hence carried out at both the regional scale (i.e.…”

Sex, size and timing: Sampling design for reliable population genetics analyses using microsatellite data

“…RNAse treatment was applied after resuspension in 20 μl of ultrapure water. Amplification of 16 microsatellite markers was conducted with QIAGEN ® Multiplex PCR kits as described in Vandewoestijne et al (2012). The genotypes were scored using Genemapper ® 3.7 (Applied Biosystems ® ), and manually verified to correct for automatic scoring errors when necessary.…”

Characterization of ten polymorphic microsatellite markers for an endangered butterfly Argynnis niobe and their cross-species utility in the closely related species A. adippe (Lepidoptera: Nymphalidae)

“…These similarities are generated by recombination mediated events, such as unequal crossingover or gene conversion and through transposition of mobile elements ( Van't Hof et al, 2007). These problems have now been resolved thanks to next-generation sequencing methodology, which allow the rapid identification of large numbers of bioinformatically variable loci, without the necessity of laborious and costly cloning (Vandewoestijne et al, 2012).…”