Identification of 18 polymorphic microsatellite loci in the spruce bark beetle Ips typographus ( Coleoptera : Scolytidae ) using high-throughput sequence data

Novel microsatellite markers for the spruce bark beetle (Ips typographus L.) are identified and characterized using next generation sequencing technology. 18 polymorphic loci were obtained by screening 10,684 reads and tested on 35 bark beetle samples from different locations in Bavaria, Germany. Allelic richness ranged from two to 38 alleles, observed heterozygosity from 0.03 to 0.66 and expected heterozygosity from 0.08 to 0.97. Four loci showed significant deviation from the Hardy-Weinberg equilibrium and no linkage between loci was detected. The 18 loci, along with another six loci previously described, provide effective analytical tools for analyzing the fine-scale genetic structure of bark beetle populations. The result of this study demonstrates that next generation sequencing technology is a valuable method for isolating microsatellites of a coleopteran species. 169 * Corresponding author. 2004) was used to test the data set for genotyping errors and the frequency of null alleles. RESULTS AND DISCUSSION Eighteen loci were polymorphic with 2 to 38 alleles, averaging 5.9 alleles per locus. Their characteristics and amplification conditions are described in Table 1. Observed heterozygosity values ranged from 0.029 to 0.657, and those for expected heterozygosity from 0.083 to 0.969. No linkage was detected between loci pairs across populations in the I. typographus samples tested (all adjusted P values > 0.083). The test for HWE for each locus in each population revealed eight deviations in four loci after Bonferroni correction (Table 1). MICROCHECKER did not detect any genotyping error (e.g. scoring error) among the data set, although possible null alleles were likely at two loci (ITY12 and ITY22) resulting in the significant deviation from HWE. However, tests for HWE have a low power when sample sizes are small (Selkoe & Toonen, 2006). This work provided 18 new polymorphic microsatellites for I. typographus. The six loci previously published (Sallé et al., 2003; Stoeckle et al., 2010) together with a set of the most informative loci described above provide an effective analytical tool for genetically identifying individuals and their offspring. The major difficult encountered in developing microsatellites for Coleoptera and Lepidoptera using traditional methods is the repeated isolation of identical motifs (Arthofer et al., 2007; Meglecz et al., 2004). This is not a problem if the next generation sequencing technology in combination with the method of data screening described above is used. This technique can be used to identify microsatellites for species for which the conventional methods may be unsuccessful. ACKNOWLEDGEMENTS. We would like to thank M. Bauer, H. Saar, A. Holzwarth and T. Käser for their contributions to this study. This study was supported by a grant from the Bavarian State Ministry of the Environment and Public Health (UGV 06070204028). 170 0.114 0.101 169–173 2 3.0 55 F: CTGTTATTCTTAAGAGCCCATC R: ACACAGTGAGCAAAATACGA (CTGT)6 GU989620 ITY50 0.367 0.429 174–220 4 3.0 60 F: AAAAGTGCAACGTTCTTAGC R: TAATGGGTTATCGAGTGGTG (ATCT)5 GU989619 ITY44 0.227 0.257 227–236 2 3.0 55 F: TCTCAATTAGCTAGACGAAACA R: TTTGCTGACTTTGATTTGAA (ATT)5 GU989618 ITY41 0.414 0.057 221–224 2 3.0 54 F: TGTGTTCCATCAGCACAACA R: CGTTATACCGGTTATTATGCCTTG (AAT)7 GU989617 ITY33 0.273 0.029 324–333 3 3.0 60 F: CAACGTTGCCGCATATTTTT R: CCTGGATGGATGACAATTCC (ACT)4 GU989616 ITY30 0.161 0.171 204–213 3 3.0 52 F: TAAAGCAAGAGGGAGGCAGA R: TTCCCTTACTCTGAATTGTCACC (AAG)4 GU989615 ITY29 1 0.697 0.571 182–192 6 3.0 60 F: GCATATTTACCCCTCGGAAT R: CCCACGGAGGTTTTGGAG (AG)8 GU989614 ITY28 2 0.969 0.657 120–540 38 1.5 60 F: GCGCGACG TGCATTATTC R: AACCAATCACAAACCATGAACA (AACCT)27 GU989613 ITY26 0.560 0.457 220–229 4 3.0 57 F: GAAAAATTGACTGATTGGAAACG R: AAGCTGTTAACGCAGTGACG (ATT)4 GU989612 ITY25 0.385 0.229 187–196 4 3.0 60 F: CCTACCGGCAAGAATCAAAG R: GCGTACGTCACGACCTACCTA (ATT)7 GU989611 ITY23 3 0.820 0.457 131–194 15 1.5 57 F: GACGTTGCTTTGTGATCGTG R: ATGGCAATTACGGACCTGAA (ATT)22 GU989610 ITY22 0.595 0.314 155–161 4 3.0 60 F: CGAGTTGACAAGTCAAGCAAA R: ATCACACGCAATCGCAAC (AG)6 GU989609 ITY19 0.115 0.143 202–213 3 1.5 60 F: TTGATCCACACGGCAATAAA R: CGGTAAGTTGCTTCCAGAGC (AAG)5 GU989608 ITY16 0.269 0.314 182–185 2 3.0 60 F: CAAGTAATGGTCAAGATCAAGTCA R: GTATGGTTAGGGGTGCGATG (AAT)5 GU989607 ITY14 2 0.674 0.486 203–213 7 3.0 60 F: ATTTAGGGAAAAATCGCTGA R: TTTTAAAACAGCCTCTCTGTC (ATT)5 GU989606 ITY12 0.373 0.143 174–177 2 3.0 60 F: TCAACCATCAGTTTACTTGT R: CTTATATTTGGGTGCCATGT (ATC)5 GU989605 ITY11 0.163 0.171 256–277 4 3.0 60 F: CGTAGGCAAATCAAAAGTAGA R: CCTGAAAATAGTGCGAAATTA (GAT)7 GU989604 ITY10 0.083 0.086 175–177 2 3.0 60 F: CATGCATAAAACAGTTCGTT R: CGCACTAGCGCTTCTATTTA (GT)8 GU989603 ITY8 HW HE HO Allelic size range (bp) NA MgCl2 Ta (°C) Primer sequences (5’–3’) Repeat motif GenBank Accession no. Locus TABLE 1. Characteristics of 18 microsatellite loci of the spruce bark beetle I. typographus L.: locus designation, GenBank Accession no., repeat motif (referring to 454 sequence data), primer sequences, annealing temperature (Ta), MgCl2 concentration, number of observed alleles (NA), allele size range (including length of M13 primer), level of observed (HO) and expected (HE) heterozygosity per locus and number (bold) of populations that deviated from Hardy-Weinberg equilibrium.


INTRODUCTION
In European forests the bark beetle, Ips typographus L., is a serious pest of Norway spruce (Picea abies).In order to improve the management strategies for pest species it is important to have a detailed knowledge of their population structure, mode of dispersal and reproductive characteristics.The six microsatellites for I. typographus cited in the literature (Sallé et al., 2003;GQ372845) indicate that this species is very good at dispersing (Sallé et al., 2007).The aim of the present study was to identify additional polymorphic loci for I. typographus in order to study its fine-scale molecular genetic structure based on the identification of individuals (within infested trees and nests).As several attempts to isolate microsatellite markers for I. typographus using traditional methods have failed (Sallé et al., 2003) it was decided to use the output of a next generation sequencing platform to identify new microsatellite markers.This new technology was previously successfully used to identify microsatellite loci for two hymenopteran species (Sirex noctilio; Solenopsis invicta) (Santana et al., 2009;Ross et al., 2010).To our knowledge no microsatellites for a coleopteran species developed directly from randomly amplified genomic DNA using a high-throughput sequencer have been previously reported.

MATERIAL AND METHODS
Genomic DNA was isolated from ten bark beetles according to the method of Hogan et al. (1994), following standard phenol-chloroform extraction.The DNA was subjected to 454 sequencing at the GS-FLX LAB of Eurofins MWG Operon (Ebersberg, Germany).1/16 plate generated 10,684 reads (3.1 Mb) with an average length of 228 bp.A single Fasta file containing all reads was screened for di, tri-, tetra and pentanucleotide repeats using MSATCOMMANDER (Faircloth, 2008) with at least four repeats except for dinucleotides (6).269 loci suitable for primer design with Primer3 software (Rozen & Skaletsky, 2000) were detected.Loci with non-desired properties were ignored as described by Opgenoorth (2009).Subsequently, the Primer sequences flanking the remaining loci were transferred into an Access © database (Microsoft Access 2007) and sorted by similarity in order to avoid the repeated selection of the same loci.In addition, the sequences were aligned by using GENEIOUS v4.7 (Drummond et al., 2009) for the same reason.Out of 135 potential loci 60 primer pairs flanking 25 di-, 25 tri-, nine tetra-and one pentanucleotide from Biomers (Ulm, Germany) were randomly ordered.The fluorescent labelling was done according to Schuelke (2000) using the universal M13 Primer.From the 60 primer pairs tested on three DNA samples of I. typographus, amplified products of expected size with low rates of intense stuttering were obtained for 24 of the 60 candidates.
For genotyping, 35 individual bark beetles originating from five regions (populations) in Bavaria (Germany) were used.DNA extraction was done using the method described previously.Polymerase chain reactions (PCRs) were performed in a total volume of 15 µl with the following components: 25 ng of genomic DNA, 0.2 µM of each reverse primer and the M13 universal primer (fluorescently labelled with 6-FAM), 0.05 µM of each forward primer, 0.2 mM of each dNTP (Solis BioDyne), 1.5-3 mM MgCl2 (Table 1), 1× PCR buffer (Solis BioDyne) and 0.5 U Taq DNA Polymerase (FIREPol ® , Solis BioDyne).For PCR amplification a thermal cycler (Mastercycler Gradient, Eppendorf, Germany) was used with the following PCR profile: initial denaturation at 94°C for 5 min, 30 cycles of 30 s at 94°C, 30 s at the primer specific annealing temperature (Table 1), 45 s at 72°C, followed by eight cycles of 30 s at 94°C, 45 s at 53°C, 45 s at 72°C and a final elongation step at 72°C for 10 min.PCR products were separated on 6% polyacrylamide gels on an ABI Prism 377 automated sequencer (Perkin Elmer) and scored by reference to a ROX standard (79-540 bp) by GENESCAN ® 3.1.2and GENOTYPER ® 2.5 software (Applied Biosystems, Foster City, CA, USA).GENEPOP 4.0 (Rousset, 2008) was used to generate allele frequencies, to calculate expected and observed heterozygosities (HE, HO), and to test for linkage disequilibrium and deviations from Hardy-Weinberg equilibrium (HWE).MICRO-CHECKER 2.2.3 (Van Oosterhout et al., 2004) was used to test the data set for genotyping errors and the frequency of null alleles.

RESULTS AND DISCUSSION
Eighteen loci were polymorphic with 2 to 38 alleles, averaging 5.9 alleles per locus.Their characteristics and amplification conditions are described in Table 1.Observed heterozygosity values ranged from 0.029 to 0.657, and those for expected heterozygosity from 0.083 to 0.969.No linkage was detected between loci pairs across populations in the I. typographus samples tested (all adjusted P values > 0.083).The test for HWE for each locus in each population revealed eight deviations in four loci after Bonferroni correction (Table 1).MICRO-CHECKER did not detect any genotyping error (e.g.scoring error) among the data set, although possible null alleles were likely at two loci (ITY12 and ITY22) resulting in the significant deviation from HWE.However, tests for HWE have a low power when sample sizes are small (Selkoe & Toonen, 2006).This work provided 18 new polymorphic microsatellites for I. typographus.The six loci previously published (Sallé et al., 2003;Stoeckle et al., 2010) together with a set of the most informative loci described above provide an effective analytical tool for genetically identifying individuals and their offspring.
The major encountered in developing microsatellites for Coleoptera and Lepidoptera using traditional methods is the repeated isolation of identical motifs (Arthofer et al., 2007;Meglecz et al., 2004).This is not a problem if the next generation sequencing technology in combination with the method of data screening described above is used.This technique can be used to identify microsatellites for species for which the conventional methods may be unsuccessful.1. Characteristics of 18 microsatellite loci of the spruce bark beetle I. typographus L.: locus designation, GenBank Accession no., repeat motif (referring to 454 sequence data), primer sequences, annealing temperature (Ta), MgCl2 concentration, number of observed alleles (NA), allele size range (including length of M13 primer), level of observed (HO) and expected (HE) heterozygosity per locus and number (bold) of populations that deviated from Hardy-Weinberg equilibrium.