Samples and DNA extraction

Adult C. ciliata samples were collected in 2010 from Guiyang, Guizhou Province and Nanjing, Jiangsu Province, China. Samples were identified and preserved in 100% ethanol, and then stored at -20 °C. Genomic DNA was extracted from individual samples using Axy-Prep Multisource Genomic DNA Miniprep Kit and stored at -20 °C until needed for PCR.

Isolation of Microsatellite Markers

We used the FIASCO (fast isolation by AFLP of sequences containing repeats) method with slight modifications to develop an enriched library of microsatellite loci (Zane et al. 2002). The genomic DNA was first digested with the restriction enzyme MseI (BioLabs, Beijing, China) and ligated to MseI AFLP adaptor (5′-TACTCAGGACTCAT-3′/5′-GACGATGAGTCCTGAG-3′) in a total volume of 25 µL containing 250 ng of genomic DNA, 1 × OnePhorAll buffer, 5.0 mM DTT, 50 µg/mL BSA, 1.0 µM adaptor, 200 µM ATP, 2.5 U of MseI (NEB), and 1.0 U of T4 DNA ligase (Promega). The reaction was then incubated at 37 °C for 3 h. The digestion-ligation products was diluted (1:10) and amplified with adaptor-specific primers (5′-GATGAGTCCTGAGTAAN-3′, MseI-N) in 20 µL reactions containing 1 × PCR buffer (10 mM Tris-HCl, pH 9.0, 50 mM KCl), MgCl2 1.5 mM, dNTPs 250 uM, MseI-N 0.5 uM, 1 U of Taq DNA polymerase (TaKaRa, Dalian, China) and 5 µL diluted digestion-ligation DNA. The PCR conditions were 5 min at 94 °C followed by 26 cycles of 30 s at 94 °C, 1 min at 53 °C, 1 min at 72 °C with a final extension time of 10 min at 72 °C. After denaturation at 95 °C for 5 min, the PCR products were hybridized for 1 h at 68 °C with biotinylated (GA)₁₂, (GT)₁₂, (CA)₂₅ and (TG)₁₈, respectively. The DNA fragments hybridized to biotinylated probes were selectively captured by streptavidin coated magnetic beads (Streptavidin Magnesphere Paramagnetic Particles, Promega, Shanghai, China). The microsatellite-enriched DNA fragments were purified using PCR Cleanup Kit (Axygen,  www.axygen.com)and then amplified by MseI-N primers for the following PCRconditions: 3 min at 95 °C followed by 26 cycles of 30 s at 95 °C, 30 s at 53 °C, 45 s at 72 °C with a final extension time of 30 min at 72 °C. The purified PCR products were ligated into pGEMT Easy vectors (Promega,  www.promega.com) and then transformed into Escherichia coli strain (DH5α). The positive clones were identified by PCR using M13 primers and visualized by agarose gel electrophoresis. All positive clones were sequenced in both directions using the BigDye Terminator Sequencing Kit (Applied BioSystems) and the ABI 3730XL Genetic Analyzer (PE Applied Biosystems, San Francisco, California, USA) with 2 vector-specific primers and internal primers for primer walking. Microsatellite sequences were identified by software SSRHunter 1.3 (Li & Wan 2005). Primer pairs for each microsatellite locus were designed by Primer Premier 5.0 software ( http://www.premierbiosoft.com/primerdesign/).

PCR Amplification and Genotyping

Each primer pair was screened against 48 individuals of C. ciliata. Three kinds of fluorophores (FAM, HEX and TAMRA) were tagged with forward primers at the 5'end of each pair. PCR amplifications were conducted in 25 µL volumes including 1 × PCR buffer (10 mM TrisHCl, pH 9.0, 50 mM KCl), 1.5 mM MgCl2, 200 FM of each dNTPs, 50 ng genomic DNA, 0.75 U of Taq DNA polymerase (TaKaRa), 4 pmol of each primer. Conditions for PCR amplification were as follows: an initial denaturing at 94 °C for 4 min, followed by 42 cycles of 50 s at 94 °C, 50 s at 51–58 °C depending on the primer pair (Table 1), and 1min at 72 °C, followed by a final extension for 10 min at 72 °C. The PCR products of 3 fluorophores (FAM, HEX and TAMRA) were mixed in a ratio dependent on the brightness of bands visualized by agarose gel electrophoresis, respectively. The PCR products were run on an ABI 3730XL DNA sequencer and the electropherograms drawn through Gene Scan 4.0 were used to extract DNA fragment sizing details using Gene Mapper 4.0 software by Sangon Biotech (Shanghai) Co., Ltd.

Table 1.

Characteristics of nine microsatellite loci of Corythucha ciliata: Genbank accession number, repeat motif, allele size range, annealing temperature (ta), number of alleles (N_a), oberserved heterozygosity (H_o), expected heterozygosity (H_e) and value from the test from the exact test for the hardy-weinberg equilibrium (p-hw).

Fig. 1.

Mutational events of Corythucha ciliata clone microsatellite sequences. A and B are the first type of mutational pattern; C is the second type of mutational pattern.

Statistical Analysis

The number of alleles (N_A), observed heterozygosity (H_O), expected heterozygosity (H_E), and polymorphism information content (PIC) were calculated for each locus using Cervus version 3.0 (Marshall et al. 1998). The Hardy-Weinberg equilibrium (HWE) and genotypic linkage disequilibrium between pairs of microsatellites were tested by Genepop 3.4 (Raymond et al. 1995). Null allele frequencies were measured by MICRO-CHECKER 2.2.3 (van Oosterhout et al. 2004). All P-values were adjusted for multiple tests using the sequential Bonferroni method (Rice 1989).

Characteristics of Microsatellite Loci

Thirty five primers were designed based on 163 clones that contained a microsatellite sequence, but 9 microsatellite loci showed high polymorphisms (PIC > 0.5) (Table 1). The number of alleles per locus ranged from 2 to 13, with an average of 6 alleles per locus. The observed (H_O) and expected (H_E) heterozygosities varied from 0.146 to 0.958 and 0.290 to 0.849, respectively, in Guiyang population. The observed (H_O) and expected (H_E) heterozygosities varied from 0.483 to 0.739 and 0.443 to 0.865, respectively, in Nanjing population. The null allele frequency was lower than 0.1 in at least 1 population. After sequential Bonferroni corrections, 2 loci (CA15 and GA365) showed significant deviations from the Hardy-Weinberg equilibrium (HWE) in Nanjing population. This phenomenon may be caused by the Wahlund effect, the effect of evolutionary pressure during the process of invasion or the existence of a null allele. Loci CA200&GT26, GT26&TG100, TG100&GA365 showed significant linkage disequilibria (LD) in the Guiyang population (P < 0.01), and loci GT26 and GA5 (P < 0.01), showed significant linkage disequilibrium in Nanjing population. This phenomenon may be caused by genetic drift of the C. ciliata population after invasion. Consequently, the 9 loci can be useful for population genetics studies and explore invasive routes of C. ciliata.

Mutations of Microsatellite Sequences

In total, we obtained 1,385 bacterial colonies from the enriched library of microsatellite loci that used the biotinylated probes: (GA)₁₂, (GT)₁₂, (CA)₂₅ and (TG)₁₈. Two hundred and four recombinant clones were sequenced, but only 163 were successful. We analyzed these microsatellite sequences to determine the mutational model. We found 2 types of mutational events that could have generated the new alleles. First, the differences in numbers of repeat motifs caused size variation of alleles (Figs. 1A and 1B). Secondly, 2 different repeat motifs contributed to the allele-size variation (Fig. 1C). However, slippages in the repeat motif and flanking region were the main mutation mechanism for the newly developed microsatellites.

The findings of this study will allow us to elucidate genetic structure of the C. ciliata populations in China and in putative regions of their origin, and thereby investigate the probable route by which the pest reached China, as well as its subsequent spread in China.