Population Genetic Structure Of Coconut Leaf Bettle Brontispa Longissima (Gestro) Based On SNP And SSR Markers In China

The coconut leaf beetle Brontispa longissima（Gestro）,native to Indonesia and Papua New Guinea,is a quarantine forestry pest that has serious harm to palm plants such as the coconut Cocos nucifera L.At present,the main research on the coconut leaf beetle involve invasion and quarantine,biological characteristics,ecological characteristics,control measures,etc.However,there is still a lack of systematic research on the population genetics of this invasive pest.In this study,the SNP molecular markers were screened by RAD sequencing and the population genetic differentiation of B.longissima in China was studied based on these markers.In the meanwhile,a rapid method of developing SSR polymorphic loci was established based on transcriptome data,and the population genetic structure and diversity of B.longissima were analyzed using these SSR loci.This study aimed to reveal the molecular mechanism of genetic differences among the geographic populations of B.longissima,with the hope of providing a theoretical basis for the invasion mechanism of B.longissima and some new ideas and approaches for the effective and sustainable control of B.longissima population outbreak.The main findings are as follows:1.SNP screening by using RAD sequencing techniqueAs a non-model organism,a large number of single nucleotide polymorphism markers（SNPs）by restriction-site associated DNA sequencing（RAD-seq）can be obtained for B.longissima,which is a simple and rapid method to obtain molecular markers.Since there is no reference genome for B.longissima,it is not possible to annotate the SNP loci.In this study,the RAD sequenced clean data were further rigorously screened under the following conditions:（1）at least 20 individuals contained the locus where the SNP was located;（2）the minor allele frequency（MAF）is0.01;（3）the minimum depth of the locus where the SNP is located is 7;（4）if the deletion rate of all samples at this locus is more than 10%,the locus is removed.A total of 10127 high-quality SNPs were obtained.These new SNP markers will serve as valuable tools for future research on the population and conservation genetics.2.Genetic differentiation of different geographical populations of B.longissima based on SNPBased on the obtained 10127 SNPs,phylogenetic tree construction,population genetic structure analysis,principal component analysis and F_STanalysis of B.longissima from different geographical populations in China were carried out.The results showed that all population samples were divided into Hainan population and non-Hainan population.The Hainan population was relatively stable,and the non-Hainan population differentiated into smaller subpopulations.At the same time,the present study analyzed the optimal K value of sample clustering,and the results indicated that K=4 was the most suitable clustering number.Principal component analysis results further verified the above conclusions.According to the analysis results of genetic structure and phylogenetic tree,it could be speculated that Taiwan and Hainan maybe the two invasive base points of B.longissima with Taiwan population invading Fujian,Guangdong,Guangxi and Yunnan（Xishuangbanna）,and Hainan population invading Yunnan（Ruili）and Sansha.3.SSR development in B.longissimaIn this study,SSR loci were searched from the transcriptome data of B.longissima collected from Xiamen,Zhangzhou and Fuzhou using the MIscro SAtellite software,1526,2011 and 1982 SSR polymorphic sites were identified in Xiamen（Group A）,Zhangzhou（Group B）and Fuzhou（Group C）respectively.The analysis script was then set up for mixed analysis regarding Contig sequence information and Unigene sequence information from these three samples,the results discovered 7 SSR site sequences that were different in the three sets of sample transcription groups.Through primer design and verification by PCR experiments,there were 6 SSR sites with good polymorphism,which could be used for the analysis of genetic structure and genetic diversity of B.longissima.4.Genetic diversity and structure analysis of B.longissima populations based on SSR markersA total of 468 samples were collected from 18 geographical populations in Hainan,Taiwan,Guangdong,Guangxi,Yunnan and Fujian province in China,the genetic diversity and structure of these 18geographical populations were analyzed by using 6 SSR loci developed in this study.The results showed that all populations had genetic diversity,16of which deviated from Hardy-Weinberg equilibrium（HW P-Val）（P<0.001）,and 11 populations showed inbreeding phenomenon.The population differentiation index F_ST reflected the low degree of differentiation among different geographic populations of B.longissima in China,which was consistent with the genetic differentiation results of B.longissima based on SNP in this study.The results of Bayesian clustering analysis based on SSR indicated that all populations were clustered into 3branches,the Ruili Population was clustered separately,and the genetic structure of Hainan population（except Ledong and Sansha）was similar,and the remaining populations showed no obvious regularity.The correlation analysis of genetic distance and geographic distance based on SSR found that there was a correlation between the 18 geographic populations of B.longissima and their genetic distance（r=0.378,P<0.05）.