Population Genetic Structure Analysis Of Tetranychus Truncatus By Microsatellites And Mitochondrial DNA

The spider mite Tetranychus truncatus Ehara,belonging to Arachnida,Acari,Acariformes,Tetranychidae,is a very important phytophagous mite pest.It is now one of the main agricultural and economic crop pests in East and Southeast Asia.Like many other Tetranychus species,T.truncatus do harm to the crops with the piercing-sucking mouthparts.Its stylet pierces into the tissue of plant and feed on the chloroplastid and cytosol of palisade cells.T.truncatus causes enormous damage on cotton plants and maize by making their leaves fall.Tetranychus urticae,a close species of T.truncatus,is the most studied mites.The genome of T.urticae was published at the end of 2011.According to five years of investigation of our lab,we found that T.truncatus have a trend of becoming the main pest mites.However,genetic studies of Chinese populations of T.truncatus have been hampered by a lack of polymorphic molecular markers especially microsatellite markers.Wolbachia is maternal inherited endosymbiont that infect most arthropods.It can manipulate host reproduction by cytoplasmic incompatibility(CI),feminization,thelytokous parthenogenesis and male killing,which contribute to the stable and spread of Wolbachia in hosts.Considering the specific properties of Wolbachia,many scientists suggested that Wolbachia may have complex impact on the diversity and genetic structure of host.Sometimes,it even leads to speciation.The reports of Wolbachia in spider mites are accumulated.However,the effect of Wolbachia on the diversity and genetic structure of host is still unknown.To address the above issues,we used the microsatellite markers and mitochondrial COI gene to investigate the diversity and genetic structure of T.truncatus populations in China.Moreover,we tried to find the relationship between divergence and geographic distance/temperature/Wolbachia.Due to the lack of SSR markers,first we combined aedeagus morphology and PCR-RFLP technology to identify T.truncatus accurately from its close mites such as T.urticae,T.phaselus,T.ludeni and T.piercei.After that,taking the advantage of transferability of SSR,we cross amplified the SSR markers screened from the genome of T.urticae in T.truncatus.Then,a cost-effective high-throughput genotyping method was developed and tested in T.kanzawai,Frankliniella occidentalis and Nilaparvata lugens.Finally,we used SSR markers and the genotyping method we developed plus COI gene to study the genetic diversity and structure of Chinese T.truncatus populations.Some former studies showed that more than 30%sequence of spider mites in GenBank were misidentified,which made sole molecular identification unreliable.Meanwhile,the morphology of T.truncatus is very close to T.urticae,T.phaselus and T.ludeni etc.Moreover,morphology identification is not suitable for high-throughput experiments.Therefore in this study,we confirmed the identification of T.truncatus by both aedeagus morphology and PCR-RFLP(restriction fragment-length polymorphism)technology.This not only laid a solid foundation for the following experiments but also clearly identified T.urticae whose genome is precious resources for SSR isolation.Besides,the identified T.kanzawai was used for testing the robustness of genotyping method.Nowadays,a large number of non-model organisms are short of available SSR markers since SSR development is time-consuming and high-budget.Therefore,population and other studies of T.truncatus have been impeded by the lack of microsatellite marker.Previous studies indicated a high potential of cross-amplification of microsatellites in Tetranychus species,meaning that the microsatellite flanking sequences are sufficiently homologous among Tetranychus species that the primers for one species may work in another species.Here,we tested 205 primer pairs designed from the whole genome sequence of T.urticae,a sister species of T.truncatus,for microsatellite makers in three populations of T.truncatus in China.About half(102)of these primer pairs yielded the desired PCR products,36 of which revealed polymorphism in T.truncatus.Each of the 36markers harbored between 2 and 23 alleles,with a mean polymorphic information content(PIC)of 0.589(0.119-0.922 range).The mean observed and expected heterozygosity across loci and the three populations were 0.063-0.844 and 0.061-0.903 respectively.Of the 36 primer pairs,22 of them also worked in Tetranychus piercei,but only a few of them worked in T.ludeni and T.phaselus.Cross-amplification is thus a cost-effective way to develop microsatellite markers,which can be of great value in population genetics studies.Considering that the number of samples and loci are large in this study,the conventional genotyping(directly fluorescence labeled)is too much expensive.The cost of fluorescence and thousands of repetitive PCR process are the main limitations.In order to solve the problem of genotyping,four new universal primers were developed.Multiplex PCR were modified from former three primer genotyping system.Each PCR contains one modified forward primer(universal primer sequence added to 5'),one usual reverse primer and one fluorescent-labeled universal primer.The three primer system was improved by simultaneous use of four universal primers combined with four fluorophores to genotype8-12 loci in one single run.The multiplex universal primer three-primer approach can dramatically reduce the cost when genotyping the microsatellites.This multiplex genotyping method was tested on T.truncatus XZ population.To further test universality,published microsatellite loci of T.kanzawai,Frankliniella occidentalis and Nilaparvata lugens were modified as needed for test.The robustness of the method was confirmed by comparing with singleplex using multiple fluorophores and genotyping two populations of T.truncatus.This method showed lower signal strength than the singleplex three-primer system,but it was still sufficient to determine the fragment length which remained consistent as before.We genotyped a total of 1159 individuals from 22 T.truncatus populations in China by22microsatellite loci and COI gene.Compared with other Tetranychus species,the genetic diversity and population divergence of T.truncatus were relatively high(Global FST = 0.21,P<0.001)except JQ and JMN.By Bayesian clustering,22 populations were divided to two clusters and the components of these two clusters varied according to the temperature zones.There was an also mild IBD effect among T.truncatus populations,we suggested that geographical distance and temperature played main role in population differentiation.When we used COI gene to investigate the mitochondrial genetic structure of T.truncatus,19 mitochondrial haplotypes were found.The haplotype diversity and nucleotide diversity were relatively high among T.truncatus populations except JMN,JQ and XZZ where only one haplotype was found.Infection rate of Wolbachia ranged from 2.82%of ZJ to 80.39%of BTB.According to wsp gene,we found Ori and Con strain of Wolbachia.Ori strain was widespread among populations(except JMN)while Con strain was limited to JMN,ZZ,BTB and BTC.Superinfection was found mainly in BTB and BTC.Even if there was no consistent one-to-one match between Wolbachia and COI haplotype,Wolbachia played a significant role in COI divergence.This results help to understand the relationship between Wolbachia and host population structure.By comprehensive analysis the genetic structure and Wolbachia infection status,we concluded that the nuclear genetic divergence of T.truncatus are positive correlated with geographical distance and closely related to the temperature zones.The genetic divergence had little relationship with Wolbachia.Our results are important for understanding outbreak mechanism and the pest management of T.truncatus.