Development Of Microsatellite Primers And Population Genetics Based On High-throughput Sequencing

Microsatellites are DNA sequences that consist of tandem repeats of 1-6 nucleotides.As a molecular marker system with high polymorphism,they provide abundant genetic information for studying population genetic structure and genetic diversity.Trilophidia annulata Thunberg 1815 is a species of locust within the family Oedipodidae(Orthoptera),and is widely distributed in China and some adjacent areas except for Qinghai-Tibet Plateau,occurring in diverse environments.However,little is known about its intraspecific differentiation.In this study,high-throughput sequencing technology was employed to analyze the microsatellite information of T.annulata,and a set of polymorphic microsatellite loci was characterized.Moreover,population genetic analysis was carried out on 18 populations of T.annulata in China.The main findings are as follows:1.Characterization of microsatellite information and development of microsatellite markersThe whole genome of T.annulata were randomly sequenced using Illumina HiSeq 2000 platform.After the de novo assembly using CLC Genomics Workbench,29,978 contigs were derived from 19,143,466 reads with the average length of 115.7 bp.A total of 1,308 potential microsatellite loci were detected from the contig collection with the abundance of 72.23 loci/Mb.Most changes of microsatellites repeats are 4-25 times,and repeat units are predominantly A and T.Based on the microsatellite sequences,45 loci were randomly sampled and used for the marker development;a total of 27 loci with desirable amplification were identified by agarose gel electrophoresis.In all,nine polymorphic loci were identified by capillary electrophoresis.2.Population genetic assay of T.annulataThe nine polyrmorphic loci were employed to analyze the population genetic diversity and structure of T.annulata.The results are as follows:(1)A total of 71 alleles were amplified from nine microsatellites loci,and the number of alleles per locus varies between 3 and 24 with an average of 7.889 per locus.Polymorphism information content(PIC)ranges from 0.398 to 0.892 with an average of 0.581.(2)The populations of T.annulata showed high genetic diversity.Among these populations,NL population possessed two peculiar alleles.The mean number of alleles(Na),expected heterozygosity(He)and observed heterozygosity(Ho)were 4.765,0.894 and 0.619,respectively.(3)All microsatellites conform to the Hardy-Weinberg equilibrium(HWE)in almost all populations of T.annulata(P<0.05).Based on SSM and TPM models,Bottleneck analysis revealed genetic bottleneck effects in some of the populations under a certain type of detection;only one population(MX)did not show L-type on its allele frequency distribution.(4)The differentiation coefficients of T.annulata were below 0.05(0.008-0.042),indicating low levels of among-population genetic differentiation.The gene flows among populations vary between 5.675 and 31.723.Mantel test revealed significant positive correlation between genetic and geographical distances(P<0.05).(5)Based on the Nei's genetic distance and UPGMA clustering analysis,the 18 populations of T.annulata were clustered into three clades.Especially,all populations from the north of the Yangtze River[including Shaanxi populations(CA,FP,ZS,MX&LY),Sichuan populations(GA),Ningxia populations(WZ)and Shandong populations(QD)]formed one clade.Those populations from the south of China[including Guangdong populations(HN&NL),Guangxi populations(NN),Guizhou populations(ZY)and Zhejiang populations(LH)]formed another clade.And the remaining populations[including Yunnan populations(BC,DL,LK&JH)and Hainan population(SY)]formed the third clade.(6)The grouping of populations by principal coordinates analysis(PCoA)confirmed the UPGMA analysis,and both results are in accordance with the geographic distribution pattern of the populations.(7)STRUCTURE analysis showed that 18 populations could be divided into three groups,and that each group represented a specific geographical area.The present study revealed relative high genetic diversity,low genetic differentiation,frequent gene exchanges and an isolation-by-distance(IBD)pattern among the 18 population.And the genetic structure model of the populations was obtained by different clustering methods:the northern part of the Yangtze River-the South China region-the Southwest China region.These findings could be explained by the following reasons:Firstly,T.annulata has a wide distribution in China,and stretches across tropical,subtropical and temperate.The environmental heterogeneity resulted in the occurrence of high genetic diversity and distinct genetic backgrounds in some populations.Secondly,since the eastern part of China's terrain is flat,the migration of T.annulata populations lack the obstruction of natural barriers.The strong flight capacity and increasingly frequent human activities may lead to frequent gene exchanges and cause the low levels of genetic differentiation among populations;Thirdly,the existing genetic structure model may be attributed to the communication between the populations from the northern part of the Yangtze River and the South China,and those from the southwest region and the South China region.