Isolation and characterization of polymorphic microsatellite loci in the bamboo locust Rammeacris kiangsu ( Orthoptera : Acrididae )

Twelve polymorphic microsatellite loci were developed and characterized from the bamboo locust, Rammeacris kiangsu, based on enriched genomic libraries. Analysis of 30 individuals showed that the number of alleles ranged from seven to 25 with the observed heterozygosity ranging from 0.333 to 0.767 and expected heterozygosity from 0.784 to 0.963. Test of cross-species amplification showed that some of these microsatellite markers could be used for studying other species such as Ceracris nigricornis, C. fasciata, and Chorthippus brunneus. 663 * Corresponding author. followed by phenol-chloroform extraction and ethanol precipitation (Sambrook & Russell, 2001). Of the 56 primer pairs tested, 24 successfully amplified the target regions, but only 12 of them revealed microsatellite polymorphisms. The number of alleles at each polymorphic locus, their size range, and observed and expected heterozygosities were calculated using Cervus 2.0 software (Marshall et al., 1998). The results are shown in Table 1. The number of alleles per locus ranged from 7 to 25. These microsatellite loci showed high levels of polymorphism. Total observed and expected heterozygosities ranged from 0.333 to 0.767 and 0.722 to 0.963, respectively. Deviation from HardyWeinberg equilibrium (HWE) and linkage disequilibrium at each locus was calculated using GenePop 3.4 (Raymond & Rousset, 2004). No significant linkage disequilibrium was detected among the twelve loci. After sequential Bonferroni correction (Rice, 1989), most of the loci (WJ622, WJ623, WJ625, WJ627, WJ628, WJ629, WJ630, WJ631, and WJ632) showed significant deviations from Hardy-Weinberg equilibrium. The use of Microchecker (van Oosterhout et al., 2004) indicated that this phenomenon may be the result of null alleles. However, this may also be attributable to a Wahlund effect (Wahlund, 1928). The Wahlund effect may result in heterozygote deficiency that is actually caused by subpopulation structure, and is the most likely explanation of the results presented here. In addition, the small sample size was an important reason for the low power of the tests for linkage and HW equilibriums. In order to assess interspecific amplification, cross-species amplification was tested in four other locusts, two Ceracris species and two species from the genus Chorthippus (Table 2). DNA samples from four individuals of each species were tested, using the same PCR conditions as for R. kiangsu. Nine of the twelve loci (except WJ621, WJ627, and WJ631) can be amplified successfully in at least one other species, with one loci (WJ625) amplified in all species tested. The results suggest that the microsatellites isolated from R. kiangsu can be useful, specially half of them, to conduct population genetic studies on Ceracris nigricornis, but their potential for cross-species amplification within Acrididae is limited. ACKNOWLEDGEMENTS. Our thanks are due to Y.F. Sun and S.Z. Li for their assistance during this study. This work was supported by a grant from the National Natural Sciences Foundation of China (No. 30670257). We also wish to thank to two anonymous reviewers for their valuable comments and suggestions. 664 Pop1, Chongqing population (n = 10); Pop2, Jiangsu population (n = 10); Pop3, Guangxi population (n = 8), and Guizhou population (n = 2). Ta, optimal annealing temperature; NA, number of alleles; HO, observed heterozygosity; HE, expected heterozygosity. Significant deviations from Hardy-Weinberg equilibrium after sequential Bonferroni correction at P < 0.05 are marked with an asterisk. Primer sequences with ‘M13-’ indicate M13F (-29) (5'-CACGACGTTGTAAAACGAC-3') that was added to the 5'-end of the primer. 0.821 0.400 0.732 0.500 0.784 0.800 0.788 0.600* 7 55 214–242 F: M13-GCGTGTACCCTAGTGATGC R: TACCTGCTGGGCTAATGTG (TG)5(TATG)6 (CATG)3 WJ632 GQ267829 0.900 0.700 0.537 0.700 0.821 0.600 0.786 0.667* 11 60 241–265 F: M13-GCATAGGAACGCACAGTAG R: GTAACCCCACAGCGATTG (TGTA)2TA TG(TGTA)6 WJ631 GQ267828 0.905 0.700 0.768 0.700 0.858 0.400 0.860 0.600* 10 60 185–207 F: CGCTTCTGTAGGAGCTTTCTAAC R: M13-GTCAGTCTAGGGATCGATGACC (TG)24A(GT)2 (GA)8A(AG)15 WJ630 GQ267827 0.895 0.500 0.821 0.600 0.837 0.700 0.905 0.600* 14 64 198–232 F: CGTGCTTTGGTTCATGGGGTTA R: M13-TGCTGTCACATCGGATCTTCG (GA)23 WJ629 GQ267826 0.732 0.100* 0.868 0.100 0.847 0.800 0.829 0.333* 11 62 211–245 F: M13-GAGGGACTGATGACCTTAGCA R: CGGACCCACGTAACTACAGACT (CT)16CA(CT)2 ACGG(AC)10 WJ628 GQ267825 0.884 0.500 0.926 0.600 0.816 0.600 0.898 0.567* 11 65 213–241 F: M13-CTTGAGGACGACACCGCATTG R: CTCCCAATAACCCGCATCAGA (AG)27 WJ627 GQ267824 0.947 0.600 0.963 0.800 0.816 0.700 0.963 0.700 25 59 200–256 F:M13-CGGGTTTGTAATAGATGGTTGTCC R: GGCGTGGTCTGTAATTTCAGAAG (GA)20T(AG)5 (CG)2A(AG)19 WJ626 GQ267823 0.848 0.200 0.953 0.700 0.942 0.500 0.945 0.467* 20 55 201–253 F:M13-TCAATACATCTTGCTGCTACGCG R: GCCTATCAATCACTGCCCCATC (GA)7T(AG)20 WJ625 GQ267822 0.942 0.500 0.963 0.800 0.921 0.500 0.946 0.600 19 52 166–212 F: M13-CGTTTCGTCGTCGAGGTAAAAT R: CCGCTGCTATAATGATCAAGGGA (TG)19(AG)23 WJ624 GQ267821 0.826 0.400 0.905 0.800 0.805 0.400 0.884 0.533* 12 60 257–281 F: M13-CAGTCCTTGCTCAACCGT R: CAAAGGCGGTGGCATAAT (GA)21 WJ623 GQ267820 0.847 0.500 0.753 0.500 0.889 0.800 0.860 0.600* 14 55 173–205 F: M13-GCAGGATGACCTTTTGAG R: TGCTTCGAGTCACTGTTC (GA)27 WJ622 GQ267819 0.932 0.700 0.916 0.800 0.853 0.800 0.899 0.767 16 57 243–283 F: M13-CGGAGTGTTTCGCCTTCT R: ACGGGACTCGTTTACTACCA (AG)28 WJ621 GQ267818 HE HO HE HO HE HO HE HO NA Pop3 Pop2 Pop1 Total Ta (°C) Size range (bp) Primer sequence (5'–3') Repeat motif Locus accession no. TABLE 1. Characteristics of the twelve polymorphic microsatellite loci isolated from bamboo locust. +, successful amplification; –, unsuccessful amplification. + – – – + – – + – – – – Chorthippus changbaishanensis + – – – + – – + – – – – Chorthippus brunneus – – + – – – – + – – – – Ceracris fasciata – – + – – – + + + + + – Ceracris nigricornis WJ632 WJ631 WJ630 WJ629 WJ628 WJ627 WJ626 WJ625 WJ624 WJ623 WJ622 WJ621 Locus TABLE 2. Results obtained from cross-species amplification tests on two Ceracris species and two species of the genus Chorthippus.


INTRODUCTION
The bamboo locust, Rammeacris kiangsu, is one of the most important pests of bamboos in China (Li et al., 1998;Zhang et al., 2007).The nymphs and adults of this locust mainly feed in large groups on the leaves of bamboo plants, often causing new culms to die and a decrease in the production of new shoots (Xu & Wang, 2004).Great efforts have been made to find effective strategies and methods for controlling this species (Shen et al., 2009).Here we describe the development of twelve microsatellite loci from R. kiangsu.Because of their high polymorphism and co-dominance these loci will be useful as molecular markers for determining the levels of genetic diversity in bamboo locust populations.
Then, nonspecific binding and unbound DNA was removed by several non-stringent and stringent washes.
These microsatellite-enriched DNA fragments were purified using EZ-10 Spin Column PCR Products Purification Kit (BIO BASIC INC., Shanghai, China) and then amplified for 35 cycles using MseI-N primers.The PCR products were ligated into pGEM-T Easy vectors (Promega) and transformed into Escherichia coli strain (DH5 ).Transformed cells were cultivated at 37°C for about 16 h on LB agar plate containing ampicillin, X-gal, and IPTG for blue/white selection.Insert-positive bacterial clones were amplified using M13 primers and visualized by agarose gel electrophoresis.Ninety-five positive clones were screened and sequenced.
Fifty-six primer sets were designed through Primer 3 software (Rozen & Skaletsky, 2000) and synthesized.Fluorescent dye labelling of PCR fragments was performed with three primers: a sequence-specific forward primer or reverse primer with M13 tail at its 5' end (5'-CACGACGTTGTAAAACGAC-3'), a sequence-specific reverse primer or forward primer and the fluorescent-labelled M13 primer (either IRD700 or IRD800, LI-COR).PCR amplifications were carried out in 15 µL volumes containing 50 ng template DNA, MgCl2 1.5 mM, dNTPs 250 µM, 0.75 U of Taq DNA polymerase (TaKaRa), 1×PCR buffer, 2.5 pmol of each primer and 0.5 pmol of fluorescently labelled M13 primer [either IRD700 or IRD800 (LI-COR, Nebraska, USA)].The amplifications included an initial denaturing at 95°C for 5 min, followed by 35 cycles of 30 s at 95°C, 30 s at 52-65°C depending on the primer pair (Table 1), and 30 s at 72°C, followed by a final extension step for 10 min at 72°C.PCR products were separated on 6.5% polyacrylamide gels using a LI-COR 4300 automated DNA sequencer and analysed using LI-COR saga GT software.

RESULTS AND DISCUSSION
The primers synthesized as described above were used for screening microsatellite polymorphism in 30 individuals collected from four different local populations (Jiangsu province, Chongqing municipality, Guangxi autonomous region, and Guizhou province).Genomic DNA from these 30 individuals was extracted using standard overnight proteinase K digestion, followed by phenol-chloroform extraction and ethanol precipitation (Sambrook & Russell, 2001).Of the 56 primer pairs tested, 24 successfully amplified the target regions, but only 12 of them revealed microsatellite polymorphisms.The number of alleles at each polymorphic locus, their size range, and observed and expected heterozygosities were calculated using Cervus 2.0 software (Marshall et al., 1998).The results are shown in Table 1.The number of alleles per locus ranged from 7 to 25.These microsatellite loci showed high levels of polymorphism.Total observed and expected heterozygosities ranged from 0.333 to 0.767 and 0.722 to 0.963, respectively.Deviation from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium at each locus was calculated using GenePop 3.4 (Raymond & Rousset, 2004).No significant linkage disequilibrium was detected among the twelve loci.After sequential Bonferroni correction (Rice, 1989), most of the loci (WJ622, WJ623, WJ625, WJ627, WJ628, WJ629, WJ630, WJ631, and WJ632) showed significant deviations from Hardy-Weinberg equilibrium.The use of Microchecker (van Oosterhout et al., 2004) indicated that this phenomenon may be the result of null alleles.However, this may also be attributable to a Wahlund effect (Wahlund, 1928).The Wahlund effect may result in heterozygote deficiency that is actually caused by subpopulation structure, and is the most likely explanation of the results presented here.In addition, the small sample size was an important reason for the low power of the tests for linkage and HW equilibriums.
In order to assess interspecific amplification, cross-species amplification was tested in four other locusts, two Ceracris species and two species from the genus Chorthippus (Table 2).DNA samples from four individuals of each species were tested, using the same PCR conditions as for R. kiangsu.Nine of the twelve loci (except WJ621, WJ627, and WJ631) can be amplified successfully in at least one other species, with one loci (WJ625) amplified in all species tested.The results suggest that the microsatellites isolated from R. kiangsu can be useful, specially half of them, to conduct population genetic studies on Ceracris nigricornis, but their potential for cross-species amplification within Acrididae is limited.ACKNOWLEDGEMENTS.Our thanks are due to Y.F.Sun and S.Z.Li for their assistance during this study.This work was supported by a grant from the National Natural Sciences Foundation of China (No. 30670257).We also wish to thank to two anonymous reviewers for their valuable comments and suggestions.

TABLE 1 .
Characteristics of the twelve polymorphic microsatellite loci isolated from bamboo locust.

TABLE 2 .
Results obtained from cross-species amplification tests on two Ceracris species and two species of the genus Chorthippus.