| Ectropis grisescens Warren is an important leaf-eating pest in tea plantations,which is widely distributed in various tea producing regions of China.In this study,mitochondrial COI gene combined with SSR molecular marker technology were used to investigate the genetic and evolutionary relationships among populations,and 16S r DNA high-throughput sequencing technology was used to further study the intestinal bacterial diversity and community structure of different geographical populations of E.grisescens.The main research results are as follows:1.A pair of new COI gene primer were developed to analyze the population genetic relationship of 18 populations of E.grisescens in Fujian Province.407 COI sequences were obtained,and the bases showed A/T bias（A+T:70.7%）.98 haplotypes were detected,and the number of haplotypes in different populations ranged from 5 to 26,indicating high haplotype diversity in each population.Among them,Hap＿2 has 208haplotypes exist in 18 geographic populations,suggesting that the Hap＿2is an ancestral haplotype.WS（Hd=0.803,Pi=0.00337）is currently in the mutation product period before population expansion,while other populations have experienced long-term stable evolution.The genetic diversity of 18 geographical populations was higher,the genetic differentiation coefficient(FST)was 0.24763,and the gene flow Nm was1.52.In phylogenetic trees,most individuals in a population are grouped together and a few individuals mutate.Neutral test Tajima’s D values ranged from-2.28894（MTY）to 0.19685（SC）.Neutral genes accounted for the majority of SC population,and the population was in equilibrium.2.MISA+Primer 3+TBtools was used to search and develop SSR loci in the transcriptome of E.grisescens.A total of 12239 SSR loci were found,with a density of 221.6703/Mb and a number of duplicate motifs of 59species.The number of mononucleotide was the maximum（9401,76.8118%）,and A/T was the most dominant type（9054）.A total of 202sites were identified by simulated amplification,55 sites could be stably amplified.32 polymorphic loci were identified by capillary typing.Among them,14 showed high and moderate polymorphism in Fujian Province.3.The genetic diversity of 16 populations of E.grisescens in Fujian Province was analyzed using 12 SSR loci with high amplification rates.The polymorphism information content of 12 SSR loci ranged from 0.2603to 0.6339.A total of 119 alleles were detected,and the alleles varied from3 to 15.The observed heterozygosity HO ranged from 0.282（MTY）to0.433（NJP）,and the expected heterozygosity HE ranged from 0.397（FX）to 0.510（LBT）.The HO was lower than the HE in all populations except NJP.Sixteen populations deviated from Hardy-Weinberg equilibrium at different loci.There was genetic differentiation among different populations,and the genetic diversity of SM,YX and LBT was higher.Except SM,there was more gene exchange among different populations in Nanping region.Structure clustering,PCo A and UPGMA clustering results all showed that there were significant differences between SM,YX and LBT and other.The results of AMOVA analysis of molecular variance showed that the total genetic variation of E.grisescens was mainly derived from individual genetic variation（76%）.4.16S r DNA sequencing was performed on intestinal bacteria from 9populations of E.grisescens in Fujian Province.A total of 1505852optimized sequences were obtained,with an average length of 428 bp.Notes to 16 phyla,26 classes,62 orders,103 families,and 135 genera.The top three dominant phyla were Firmicutes（94.12%）,Proteobacteria（5.10%）and Actinobacteriota（0.62%）.Top 3 dominant genera were Enterococcus（89.30%）,Wolbachia（4.38%）and Mycoplasma（4.27%）.There are no endemic species in LBT.SC had the highest richness,which was significantly different from other populations.WT had the highest diversity,while SM had the lowest richness and diversity.COG and KEGG gene function prediction results showed that intestinal flora mainly assisted the metabolism of E.grisescens,metabolizing and transporting carbohydrates and proteins.In conclusion,the genetic diversity of E.grisescens population in Fujian Province was high,and the genetic differentiation mainly occurred among individuals within the population.There were different degrees of genetic differentiation among different geographic populations,and there was no significant correlation between genetic differentiation and geographical location.The composition structure and diversity of intestinal flora of E.grisescens were similar among different geographical populations,and the functions of intestinal bacteria were mainly to assist E.grisescens in maintaining intestinal p H and digestion and absorption of tea secondary substances.Maternal inheritance pattern and Wolbachia may be the reasons for the differences between mitochondrial COI gene and SSR in diversity analysis. |
