Isolation and characterization of microsatellite loci for Rhyacionia leptotubula (Lepidoptera: Tortricidae)

Microsatellite as one of the most popular molecular markers provides a useful tool for population genetic structure analysis in insects. But few microsatellite loci have been developed for Lepidoptera. Here we isolated and characterized 9 polymorphic microsatellite loci in Rhyacionia leptotubula (Lepidoptera: Tortricidae). The number of alleles of these loci ranged from 2 to 11. Observed and expected heterozygosities ranged from 0.0000 to 0.5294 and from 0.3905 to 0.8719, respectively. Seven loci show significant Hardy-Weinberg deviations. These loci could provide insight into population genetic structure and dispersal patterns of R. leptotubula.


INTRODUCTION
The pine tip moth R. leptotubula (Lepidoptera: Tortricidae) is a serious pest of Pinus yunnanensis and P. armandii (Huang, 1987).The larvae feed primarily on needles soon after hatching, and then bore into the shoots.Repeated attacks can cause severe deformation of host trees and significant long-term growth loss.Now it is distributed widely over much of Yunnan province in China.Recent survey showed that this pest can damage about 40% of the host plants in the outbreak area, and migrate with a speed of about 5 km to other regions per year (Wu et al., 2008).The increasing population of R. leptotubula poses an ever-increasing problem of infestations.Interestingly, we found that the distributional areas of this pest are with high mounts and deep valley as geographical barriers, and its ecological environments are in large difference.In addition, its distribution in the same occurring mountain of the altitude difference can reach 1 km (unpublished data).Such adaptations to physical barriers and diversifiable ecology may have a strong impact on the genetic structure among populations.As a basic understanding of population genetic relationships of R. leptotubula essential for effective management, the genetic study of this pest is *Corresponding author.E-mail: swfclyh@126.com.critically needed.Up to data, there are no any applicable molecular markers been developed in R. leptotubula yet.We here report the isolation and characterization of 9 microsatellite loci for this species that will be used to population genetic analysis.

MATERIALS AND METHODS
R. leptotubula larvae were collected from the same pine tree in Huize County, Yunnan province, China.Its genomic DNA was isolated using standard phenol-chloroform extraction procedures (Sambrook et al., 1989).DNA fragments from 250-1000 bp digested with RsaI (Takara) were purified from agarose gels using a QIAquick Gel Extraction Kit (Qiagen), ligated to Rsa21 and Rsa25 linkers (5`-CTCTTGCTTACGCGTGGACTA-3` and 5`-phosphate-TAGTCCACGCGTAAG CAAGAG-3`), and hybridized with the two (CT)10 and (GT)10 5-biotinylated oligo-probes (Kijas et al., 1994;Franck et al., 2005).Hybridized fragments were isolated using Streptavidin Magnesphere® Paramagnetic Particles (Promega).Captured fragments were recovered by polymerase chain reaction (PCR) amplifications to increase the quantity of the resultant microsatellite-enriched eluate using Rsa21 as primer.Cycling conditions were 1 min at 94°C, followed by 25 cycles of 1 min at 94°C, 30 s at 55°C, and 1 min at 72°C, followed by a final extension at 72°C.PCR products with the size of 250-1000 bp were purified from 1% agarose gel, ligated into pMD18-T vector (Takara), and then transformed into competent DH5α, Escherichia coli (Tiangen).Resulting positive clones were randomly selected and sequenced using an ABI automated DNA sequencer model 377 (Applied  b The range of observed alleles in base pairs.Ta, annealing temperature; Na, number of alleles; HO, observed heterozygosity; HE, expected heterozygosity; P, P-value of probability test for deviation from Hardy-Weinberg equilibrium; F, forward primer sequence; R, reverse primer sequence.Na, HO, HE, and P were estimated from 23 R. leptotubula larvae collected from one host plant.Biosystems).Primer sets were designed using Primer 3 online for the sequences with at least 5 uninterrupted microsatellite repeat units.
To evaluate the polymorphism of the primer pairs designed, 23 individual larvae were used.PCR was performed in a 10 µl reaction containing 0.5 µl genomic DNA, 2.5 pmol of each primer, 0.2 mM of dNTP, 1×PCR buffter, and 0.2 unit Taq DNA polymerase (Takara).Thermal cycling conditions were as follows: predenaturing at 94°C for 2 min; 30 cycles at 94°C for 30 s, 30 s at the primer-specific annealing temperature (Table 1) and 72°C for 30 s; followed by a final extension step at 72°C for 10 min.PCR product mixture was analyzed on 6% denaturing polyacrylamide gels.DNA fragments were visualized by silver staining.Data analysis were performed using POPgene 1.32 to calculate the average number of alleles per locus, observed heterozygosity (HO), expected heterozygosity (HE), and Hardy-Weinberg equilibrium

RESULTS AND DISCUSSION
One hundred positive clones were randomly selected and sequenced.Thirty-five of them have at least 5 uninterrupted microsatellite repeat units.Primer pairs designed for them were tested on 23 R. leptotubula individuals collected from one host plant.Finally, nine polymorphic microsatellites were obtained (Table 1).The number of alleles per locus ranged from 2 to 11 (average 6.5).Average observed heterozygosities (H O ) were 0.1075 (varied from 0.0000 0.5294).O of 7 homozygous loci was 0.0000.Average expected heterozygosities (H E ) were 0.6780 (varied from 0.3905 to 0.8719), and no significant LD of them was detected, suggesting a high degree of intrapopulation variation in isolated loci.Locus Rel43 and Rel57 were detected with no significant deviation from HWE.The HWE deviation of other 7 locus is probably because of the occurrence of null alleles.In addition, R. leptotubula individuals tested in this study were collected in a sampling scheme intended to look at population genetic structure at small place.Therefore, such HWE deviations could be attributed to sampling bias as all individuals from one host plant might be originated from the same one adult female.The exact reason requires further investigation.Moreover, it is well known that isolation and characterization of microsatellite markers is clearly more difficult in Lepidoptera than in most other organisms, and very few microsatellite loci have been reported for Lepidoptera (Keyghobadi et al., 2002).However, although 7 loci show HWE deviations, these primers are currently being successfully used in the samples collected from different geographic populations.Thus, these microsatellite loci described here are suitable tools for future genetic investigations in R. leptotubula.
a Expected product size based on primer sequence of original clone.