The sycamore lace bug, Corythucha ciliata (Say) (Hemiptera: Tingididae) of North America, is an invasive pest of plane and sycamore trees (Platanus spp.) (Proteales: Plantanaceae), and has invaded many countries. To explore the population genetic structure and the invasion route by which C. ciliata reached China, we developed 9 highly polymorphic microsatellites loci by the FIASCO method. Polymorphism of the 9 loci was assessed in 48 individuals from 2 populations (Guiyang and Nanjing) in China. The number of alleles per locus ranged from 2 to 13. The observed (HO) and expected (HE) heterozygosities varied from 0.146 to 0.958 and 0.290 to 0.849, respectively, in Guiyang population. Likewise HO and HE varied from 0.483 to 0.739 and 0.443 to 0.865, respectively, in Nanjing population. Two loci (CA15 and GA365) showed significant deviations from the Hardy-Weinberg equilibrium (HWE) in Nanjing population. Moreover, loci CA200>26, GT26&TG100, and 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 disequilibria (LD) in the Nanjing population. Finally, we found 2 types of mutational events that could generate the new alleles, but the main mutation mechanism for the newly developed microsatellites was slippage in the repeat motif and in the flanking region. In future work, the nine loci identified here will be used to study the population genetic structures of C. ciliata populations in China and in putative regions of their origin, and investigate the probable route by which the pest reached China.
The sycamore lace bug, Corythucha ciliata (Say) (Hemiptera: Tingidae) is a new invasive insect species in China from North America (Halbert et al. 1998) that previously had invaded many countries including France, Germany, Chile, Korea, Japan (Wulf et al. 1987; Prado 1990; Chung et al. 1996; D' Aguilar et al. 1997; Tokihiro et al. 2003 ). In invaded locations, C. ciliata feeds primarily on leaves of plane and sycamore trees (Platanus spp.; Proteales: Plantanaceae), reducing photosynthesis and promoting diseases, which may be followed by death of the foliage.
Although the physiology and ecology of C. ciliata and its control with pesticides have been investigated (Yoon et al. 2008; Ju et al. 2010, 2011a, 2011b), its population genetics is unknown. To better control this pest, an increased understanding of its population genetics and invasion routes is needed. Microsatellites markers can help identify the origins of newly established populations of invasive species, as well as their genetic makeup and their routes of migration (Behura 2006; Ascunce et al. 2011). However, the microsatellite loci of C. ciliata have not been reported and few expressed sequence tags (ESTs) of C. ciliata are available in the public domain, hindering the study of the population genetics and routes of migration of this species. Consequently, we used the FIASCO method (fast isolation by AFLP of sequences containing repeats) with slight modifications to develop an enriched library of microsatellite loci (Zane et al. 2002) to screen polymorphic loci. Here, we present sequences of 9 microsatellite loci for C. ciliata.
Materials and Methods
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.
Characteristics of nine microsatellite loci of Corythucha ciliata: Genbank accession number, repeat motif, allele size range, annealing temperature (ta), number of alleles (Na), oberserved heterozygosity (Ho), expected heterozygosity (He) and value from the test from the exact test for the hardy-weinberg equilibrium (p-hw).
The number of alleles (NA), observed heterozygosity (HO), expected heterozygosity (HE), 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).
Results and Discussion
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 (HO) and expected (HE) heterozygosities varied from 0.146 to 0.958 and 0.290 to 0.849, respectively, in Guiyang population. The observed (HO) and expected (HE) 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>26, 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.
Special thanks to Y. Ji and J. Xu, students at Yangzhou University. This work was supported by Science & Technology Program of Yangzhou (YZ2010064) and the National Natural Science Foundation of China (31300467).