Genetic Diversity Analysis And SSR Primer Screening In Colorado Potato Beetle,Leptinotarsa Decemlineata(Say)

Corolado potato beetle(CPB),Leptinotarsa decemlineata(Say),has been identified as a main quarantine object in China.Since the invasion into China,CPB spread from west to east and now widely exists in Northern Xinjiang.To develop appropriate,effective and integrated pest control strategies,spatial distribution and propagation regularity of CPB should be further studied.Moreover,molecular genetic diversity analysis is a crucial method to solve this problem.In this study,SSR and RAPD molecular markers are used to analyze genetic diversity among CPB in different geographical populations and to locate the SSR loci in CPB's genome.By molecular markers,SSR primer is also redesigned and validated.Chief results are summarized as following: 1?When SSR molecular marker was used to study the CPB's genetic diversity,main results were classified as following:At the level of species,8.2500 alleles(Na)and 3.5552 effective alleles(Ne)were detected in CPB's genome.And the ratio of polymorphic loci was 100%.At the level of geographic population,the number of alleles(Na)was 2.3750 in Chabuchar and 4.2500 in Mulei,averaged 3.21875.The number of effective alleles(Ne)was 1.8986 in Chabuchar and 3.3169 in Bole,averaged 2.5100.And the ratio of polymorphic loci was 75% in Hutubi and 100% in Heilongjiang Province,averaged 97.5%.In addition,Nei's expected heterozygosity was 0.4125 in Chabuchar and 0.6550 in Bole,averaged 0.5438.Shannon's diversity index(I)was 0.6597 in Chabuchar and 1.2238 in Mulei,averaged 0.9505.What's more,for CPB's different populations,the inbreeding coefficient(Fis)was 0.1241,the coefficient of gene differentiation(Fst)was 0.2068,and the gene flow was 0.9588 by the use of POPGENE and SSR molecular marker.2?When RAPD molecular marker was used to study the CPB's genetic diversity,some important findings were shown as follows:At the level of geographic population,the number of alleles(Na)was 1.2143 in Bole and 1.6071 in Nilka,averaged 1.4071.The number of effective alleles(Ne)was 1.0835 in Bole and 1.1999 in Manas,averaged 1.1385.And the ratio of polymorphic loci was 21.43% in Bole region and 60.71% in Nilka,averaged 40.72%.Moreover,Nei's expected heterozygosity was 0.0561 in Bole and 0.1216 in Manas,averaged 0.0962.Shannon's diversity index(I)was 0.0907 in Bole and 0.1935 in Qitai,averaged 0.1594.At the level of species,2.0000 alleles(Na)and 1.1391 effective alleles(Ne)were found in CPB's genome.And the ratio of polymorphic loci was 89.3 %.In addition,Nei's expected heterozygosity(H)was 0.1138 and Shannon's diversity index(I)was 0.2159 ± 0.1292.Both index were relatively low,which indicated a low genetic diversity.Genetic diversity value(Ht),Hs,the coefficient of gene differentiation and the gene flow were respectively 0.1150±0.0072,0.0962±0.0044,0.1637 and 0.9588.In other words,the probability of genetic variation among different populations was 16.37%,and the probability among the same pupolation was 83.63%.Neither by RAPD molecular marker nor by SSR molecular marker,a same conclusion can be made: genetic differentiation among the same population was low.3?According to RAPD molecular marker and cluster analysis of CPB from different geographic population,genetic distance between Urumqi's CPB and Tacheng's CPB is the closest.Additionally,SSR molecular marker and cluster analysis reveal that there are mainly two CPB populations,one in Northeast China and the other in Northwest China.CPB from Qitai,Mori Kazak Autonomous County,Manas county,Fukang and urumqi county collectively constitute one population.CPB in Kazakhstan exhibits a closer genetic relationship with those in Bole region and Emin County.4?CPB's genome contains 81937 SSR luci,of which 53099 are mononucleotide,9770 are dinucletide,13951 are trinucleotide,3232 are tetranucleotide,1681 are pentanucleotide and 20 are six nucleotides.Furthermore,a large number of A and T bases are found in the sequence of the SSR loci,including A/T,AG/CT,AAT/ATT.5?19 SSR primes were randomly selected from those which were already designed to amplify CPB's DNA[DNA of CPB from 15 different geographic populations],of which only 14 can be used to amplify bands,and only 11 were polymorphic.For those 11 primers,the PIC values ranged from 0.375 to 0.794,and averaged 0.600.Therefore,they have great value in polymorphism investigation.