| Athetis lepigone(Moschler),which belongs to Lepidoptera,Noctuidae,Athetis,is distributed in Parts of Europe,Japan,Russia,China,North Korea and other places.It is a new pest of maize in China and has already started out in Shandong,Henan,Hebei,Jiangsu,Anhui,Shanxi and Beijing.The sudden outbreak of Athetis lepigone has seriously jeopardized agricultural production and posed a great challenge to plant protection.The mechanism of outbreak of this moth is not clear,there are two main views,the first is that the outbreak of this pest is triggered by rapid in situ population size increases in each outbreak area;the second is that the outbreak is triggered by immigrants from a potential source area in China.Although methods such as high-altitude capture,radar monitoring and mark-release-recapture recycling are often used to study the spread,migration and outbreaks of pests.However,these methods often require a lot of manpower,material and financial resources,and the difficulty of experimenting on small insect coefficient is large.The study of the genetic structure of the population of Athetis lepigone can not only reveal the genetic relationship between the geographical populations of the worm,but also explain the range and intensity of gene flow between populations.Compared with the traditional high-altitude network capture,radar monitoring and mark-release-recapture recycling and other methods,population genetic analysis can save time and money.At present,the molecular markers of this study are limited to ISSR and COI genes.By using transcriptome and ESTs data from NCBI,the author successfully amplified the mitochondrial genome of A.lepigone and developed COI+ND1+CYTB gene as molecular marker for population genetic structure analysis and 10 pairs of stable polymorphic microsatellite locus.The main conclusions drawn from the study on the genetic structure of the population were as follows:1)Using next-generation sequencing technology,we successfully obtained 13 complete A.lepigone mitochondrial genomes that ranged from 15,581 bp to 15,589 bp(GenBank MF152830-MF152842).We chose the shortest genome,LH39,to describe the genome organization,structure and composition in detail.The A.lepigone complete mitochondrial genome contains 13 PCGs,two rRNA genes,22 tRNA genes and a non-coding region,with an arrangement similar to those of other sequenced Noctuidae insects,and a trnM-trnI-trnQ order,which is different from the ancestral order trnI-trnQ-trnM.Of these 22 tRNAs genes,14 are encoded by the J-strand and 8 are encoded by the N-strand.The predicted secondary structures of the tRNAs are shown in.All tRNA sequences can fold into the usual cloverleaf secondary structures;however,trnSl lacks the DHU arm,which is common in insect mitochondrial genomes.As with other insect mitochondrial genomes,the arrangement of the large and small ribosomal RNA(rrnL and rrnS)located on the N-strand is conserved.rrnL is located in trnL1(CUN)and trnV,The 540-bp A+T-rich region,as the initiation of replication of mitochondria,is located between the rrnS and trnM in A.lepigone.The A+T-rich region has some conserved structures,including the motif `ATAGA’ followed by a 19-bp poly-T stretch and the motif’ATTTA’ followed by a microsatellite-like(AT)sequence.The poly-T element may be involved in controlling transcription and replication initiation.There are two long 212-bp repeat elements in the A+T-rich region of the A.lepigone mitochondrial genome.2)The results showed that sites with higher nucleotide polymorphisms were mainly-genes encoding NADH dehydrogenase and cytochrome oxidase b,such as ND1,ND4,ND5 and CYTB genes.ND2,COX2 and ATP8 genes have low polymorphism and are relatively conservative.Considering the synonymous and non-synonymous substitution rate,ω value,Tajima ’D and Fu and Li’s test results,most of the mitochondrial protein genes of the two-point moth were followed by neutral evolution.However,there are some sites where occur the insertion of the diversity of choice,mainly in the COX3,ND1,ND3,ND4,ND4L five genes.A variety of model tests demonstrated that the ND3 and ND4L genes were inserted into the candidate region of the five selected genotypes,and at least ND3 and ND4L genes were inserted into the polymorphic selection.And these genes are encoding mitochondrial complex I,suggest that the selected site and mitochondrial protein function has an important relationship.3)The traditional COI gene and other two most polymorphic genes ND1 and CYTB were selected,and three mitochondrial protein genes were used as molecular markers to study the population genetic structure of 222 individuals of 8 populations.The results showed that the genetic diversity of mitochondrial genes in 8 geographic populations was rich,and the higher populations are LH,FC,HY and XP.Neutral analysis showed that there might be background selection in LH and XP populations,while XJ populations had some recent signs of population expansion.The results of genetic differentiation index and AMOVA analysis shows there is significant genetic between SJZ lab-reared population and 7 natural poputions.The Mantel test shows that there is no geographical isolation between the 7 natural populations of Athetis lepigone.Phylogenetic analysis shows that the haplotypes of the eight geographic populations can be clustered together and can be roughly divided into two clusters,so the genetic relationships of these populations are close to each other.And populations with distant geographical distance share same haplotype indicates a strong gene flow among populations.The results show that the A lepigone has the ability to undergo a long-diatance migration.4)EST-based microsatellite development method is free of microsatellite DNA library construction and clone sequencing,which greatly reduces labor,time,and cost.The SSRs were found as molecular markers for the study of the A lepigone populations,of which 10 locus were clear and well,with an average of 6 alleles per locus.As the presence of null alleles was detected at AL2 and AL9,we deleted the two loci from the following population genetic structure analyses.For population genetic structure analysis,the 8 new microsatellites first clearly discriminated the two lab-reared populations from the remaining seven natural populations by FST,STRUCTURE,and DAPC analyses.Relatively lower levels of genetic diversity detected in the two lab-reared populations revealed that substantial genetic drift in the two lab-reared populations during rearing may have led to significant divergence.Contrast to 2 lab-reared populations,the average value of FST between pairs of the remaining natural populations was 0.000.STRUCTURE results showed that the log-likelihood of the multilocus genotypic data was maximal and with a low variance for K=1,which indicates that the seven natural populations most likely formed a single genetic cluster.BayesAss results revealed strong recent gene flow among populations.The fractions of migrants ranged from 0.0422 to 0.0531,with an average value of 0.0478.In addition,relatively higher levels of migration rate from FC,HX,and LH to other populations was found.There was no significant correlation between geographical distance and population differentiation by Mantel correlation test,so there was no geographical isolation phenomenon among A lepigone populations in China.In conclusion,the high genetic diversity of central inland areas and the ability of long-distance migration provide a genetic basis for its outbreak.The high polymorphism of the mitochondrial gene,low differentiation and strong gene flow between A lepigone populations makes us to pay more attention to the scientific and rational use of insecticides.Single kind of pesticides shoud be used cautiously to promote the resistance generated by related genes and wide spread of A lepigone. |
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