Microsatellites Isolation And Population Genetic Structure Analysis Of Tetranychus Urticae And Laodelphax Striatellus In China

Both the two-spotted spider mite Tetranychus urticae Koch and the carmine spider mite T. cinnabarinus (Boisduval), which belong to Arachnida, Acari, Acariformes, Tetranychidae, Tetranychus, are very important mite pests. The two species have multiple dispersal mechanisms. They are wingless and usually rely on crawling for their dispersal. But they can also be carried for long distance by the wind and by human activities. Due to the complex dispersal mechanisms of T. urticae, the population structure and diversity would be complex. However, due to the limited number of markers used to analyse geographic populations and individuals, the information that they provided was insufficient to understand the genetic diversity and population structure clearly. In addition to questions about the population structure and genetic diversity of T. urticae in China, there is a big question about its taxonomy. Due to reproductive compatibility, difficult to find out the differences in morphology and mitochondrial DNA levels, T. cinnabarinus and T. urticae are considered to be the single species in Europe and the United States. However, in China and other Asian countries, they are considered to be two different species of mites. The reasons are the differences of body color and reproductive incompatibility between them. Therefore, whether they constitute one or two species has been debated for long time.To address the above issues, a microsatellites-enriched DNA library was constructed using a suppression-PCR procedure. We genotyped25populations of spider mites in China using eight microsatellite loci, including2newly developed by ourselves and6previously reported. We also compared the difference of the genetic diversity and population structure of the two types of mites, and try to clarify the taxonomic status of the two types of mites from a population genetic structure perspective. The main results and conclusions are as follows:1) We successfully constructed the microsatellites-enriched DNA library of the spider mite using a suppression-PCR procedure. Of the180clones sequenced,53sequences flanking microsatellites were selected for primer design. Finally, two microsatellites with good polymorphism was isolated from the genome of the spider mite. This is the first time to use the suppression-PCR procedure for microsatellites isolation in spider mites. Low mutation rate of the mite microsatellites bring the isolation efficiency down.2) We genotyped a total of1055individuals from18T. cinnabarinus populations and7T.urticae populations in China using eight microsatellite loci. One hundred alleles were identified in the8loci, with13.63in each locus across the25populations. However, the low values of heterozygosity and allelic richness implies the low level of genetic diversity of the spider mites. In addition, genetic diversity of the red form mites was found to be higher than the green form. Pearson correlations between statistics of variation (AR and He) and geographic co-ordinates (latitude and longitude) showed that the genetic diversity of T. cinnabarinus was correlated with latitude. The declining genetic diversity of T. cinnabarinus with increasing latitude may be because the northern populations have fewer generations per year. Twenty-three of the25populations used in this study deviated from HWE, and of these,19populations displayed significant heterozygote deficiency. Further analysis revealed that the deficit in heterozygotes were due to null allele.3) We found a highly significant genetic differentiation among the25populations with global FST=0.506. Spider mites possess low levels of genetic diversity, limited gene flow between populations and significant IBD (isolation by distance) effect. These factors in turn contribute to the strong subdivision of genetic structure. Based on the results of NJ tree, Bayesian analysis and AMOVA, we conclude that five clades likely exist in China. In addition, the population genetic structures of the two forms of mites do not support their separation into two species. The morphological difference between the two forms of mites may be the result of epigenetic effects.The small brown planthopper (SBPH) Laodelphax striatellus Fallen is an important agricultural pest, which is widely distributed in temperate zones such as East Asia, Southeast Asia, Europe and Northern Africa, and can overwinter safely in these areas. This pest is one of the most serious pest insects of rice plant and many other kinds of gramineous plants. Besides the direct sucking damage, the agricultural significance of SBPH lies in its ability to spread the damaging plant disease, Rice stripe virus (RSV). Despite the economic importance of this pest, the population genetic structure and genetic diversity were little known. In recent years, due to the characteristics of high level of polymorphism, co-dominant Mendelian inheritance, high frequency of occurrence, and ease of detection by PCR, microsatellites or simple sequence repeats (SSR) have been widely used in population genetic studies. However, no microsatellites primers developed specifically for the SBPH bottlenecked the process of population genetic study for the SBPH. To resolve the aforementioned issues, we tried to develop microsatellites through searching the EST database of the brown planthopper (BPH), Nilaparvata lugens (Stal) and construction the microsatellites-enriched libraries. Employing9new microsatellites developed in this study and COI gene, we investigated the genetic structure of17L. striatellus populations in China. The main results are as follows:1) An enormous number of ESTs are now available in the public sequence database, and can be exploited to identify markers inexpensively. Compared with conventional markers derived from genomic DNA, EST-derived markers are easy to develop, and highly transferable. In this study, we mined existing BPH ESTs for new microsatellites, and validated the transferability from BPH to SBPH. Finally we developed and characterized12polymorphic microsatellites from the expressed sequence tags database of the brown planthopper (BPH), Nilaparvata lugens (Stal). Microsatellites sequencing revealed complex mechanisms of mutations of the12microsatellites loci. The fact of failed transferability of the new microsatellites from BPH to SBPH may be due to that the two species had diverged long time ago, and evolved quickly.2) Nine microsatellites loci were isolated from the genome of Laodelphax striatellus (Fallen)(Homoptera:Delphacidae) by constructing (TC)6(AC)5and (AG)6(AC)5compound SSR enriched libraries using suppression-PCR procedure. These loci developed in this study were high polymorphism with13to30alleles per locus in three tested populations. The observed and expected heterozygosities ranged from0.255to0.833and0.392to0.929respectively. These microsatellites markers can be used for population genetic structure and genetic diversity research of L. striatellus.3) The results of the microsatellites implied L. striatellus populations possess high levels of genetic diversity. The allele numbers of each locus in each population were ranged from12.111to16.333. The observed heterozygosity and expected heterozygosity were0.536-0.683and0.742-0.840respectively. The L. striatellus populations in East China have much higher levels of genetic diversity than the populations in Northeast China and South China. Most microsatellites displayed a significant departure from Hardy-Weinberg equilibrium (HWE) in most populations due to serious inbreeding. Private alleles were identified in all populations except YF and NN, which are all located in South China. This may be due to the limited number of analyzed samples in the two populations. Little genetic differentiation among almost all the populations, with global FST=0.004. However, HLJ and DD show slightly but significant differentiation with some other populations. This was also evidences by the results of NJ tree and Bayesian analysis. AMOVA results indicate that genetic variation mainly comes from the individuals’ level. Although the level of population differentiation was very low, there were also mild IBD effects among L. striatellus populations. Which means geographical distance plays a role in population differentiation.4) All sequences were truncated to the same length (769bp) to eliminate missing data. The sequences had33polymorphic sites (accounting4.2%), of which15were parsimony informative. In general, L. striatellus populations have high levels of genetic diversity. An alignment the COI gene from720individuals from17populations across China revealed37haplotypes. The haplotype diversity ranged between0.159and0.764with mean0.642. However, the nucleotide diversity was relatively low, which was ranged between0.101%and0.320%(mean0.217). The L. striatellus populations in East China and North China also showed much higher levels of genetic diversity than the populations in Northeast China and South China based on the COI gene. With5.5haplotypes per populations and no private haplotypes, the Northeast China populations posses the lowest levels of genetic diversity. Among which, HLJ was the lowest one, only3haplotypes were identified in this population. The COI also revealed little genetic differentiation among populations, although the values of FST calculated by COI gene (mean0.055) were slightly larger than by microsatellites. HLJ populations also displayed significant differentiation with some other populations according to the COI gene. AMOVA results also indicate that genetic variation mainly comes from the individuals’ level.6) According to the research on the population structure of L. striatellus by SSR and COI, we concluded that:the L. striatellus population shows a relative low level of genetic differentiation, which is similar with the migratory species Locusta migratoria manilensis (Meyen), while there is a strong gene flow among these populations indicating that the L. striatellus may have Long-distance migratory ability. Based on the COI we found that37haplotypes distribute unevenly among populations. This could be explained by the environmental selection pressures. However, whether there is a linkage between the haplotypes and the fitness, migration of L. striatellus requires further experimental verification. Furthermore, we found that individuals infected with Wolbachia don’t cause a reduction in the diversity of haplotypes but the nuclear polymorphisms, which can be reasoned by the hitchhiking.