De Novo Genomic Assemble And Sequence Analysis Of Two Pathogenic Microorganism Isolated From Antheraea Pernyi

Antheraea pernyi is a most important economic insect that primarily used for weaving silk,and also the pupa of Antheraea pernyi is of great nutrient value.Here we sequence two types of pathogen that can cause great damage to Antheraea pernyi which were microsporidia N.antheraeae and Antheraea pernyinucleopolyhedrovirus,respectively.Nosema antheraeae is the pathogen of Antheraea pernyi pebrine disease and have been found wildely in most areas raising Antheraea pernyi.Serious economic losses in recent years due to the Antheraea pernyi pebrine disease spread to Antheraea pernyi production,this pathogen has become a serious factor restricting the development of Antheraea pernyi industry.Currently,the mainly way to control Antheraea pernyi pebrine disease is quarantining N.antheraeae in silkmoth to prevent maternal infection.At present,most of the studies of Nosema antheraeae mainly focus on ecologic level instead on the genetic level.Based on the molecular tools to explore the infection mechanism of Nosema antheraeae will be helpful for establish efficient and accurate disease quarantine and control methods.The disease of Antheraea pernyi nuclear polyhedrosis always cause 30%decrease yield of the cocoon production of A.pernyi,so the A.pernyi nucleopolyhedrovirus will also brings great economic losses in the Chinese silkworm breeder.Although people have finished two complete genomes of A.pernyi nucleopolyhedrovirus,the SNP,indel and mutation among different isolates has never been referred.So it is siginificant to study of genomic variation between different strains within species,which will provide a theoretical basis for the prevention and treatment of A.pernyi nucleopolyhedrovirus.These studies firstly use bioinformatics methods in comparision with the Pulsed Field Gel Electrophoresis results to estimate the genome size of Nosema antheraeae.We finally estimate that the genome size of Nosema antheraeae is about 7M.By Comparing with the results used four de novo assember,the most optimal de novo assembly software for Nosema antheraeae genome is determined.Our results show that when Velvet assembler is used to assemble the genome(k-mer = 31),total genomic length is 5,174,210 bp and the longest contig is 461,762 bp.Meanwhile the values of N50 reach 47295 bp.All these main specifications are much better than assembly results that have been reported previously.And then,we use the phred-phrap-consed package on the best Velvet Assembly results to construct the scaffold and to fill the gaps.After that,the length of total scaffolds is 5777003 bp and N50 is 63793 bp.At the same time,the longest scaffold is 460288 bp.3188 CDS were predicted by using Glimmer software,and the total-length of all CDSs is 3.45M.The average length of CDS is 1021 bp.COG annotation were also conform,which indicate that no dinstict functional divergence between chose Nosema species.The synteny maps were drawn to show that degree of genome conservation between N.antheraeae and N.bombycis.Based on the sequencing and genomic structure analysis of Antheraea pernyi nucleopolyhedrovirus isolated from Henan Province,China,we found the whole genome size of AnpeNPV-H is 125,954 bp and G+C content is 53.4%.There are predicted 147 ORFs in genome and each ORF of is at least 50 amino acids.95 ORFs have explicit known function included 30 conserved genes reported in most baculovirus.We analysis genome-wide single nucleotide polymorphism(SNP),indel events and evolution between AnpeNPV-H,AnpeNPV-Z and AnpeNPV-Z(Two Antheraea pernyi nucleopolyhedrovirus published by Nie etc.,Fan,etc.in 2007).In total,there are 204 SNPs between AnpeNPV-H,and AnpeNPV-Z;there are 204 SNPs between AnpeNPV-H and AnpeMNPV-L;there are 175 SNPs between AnpeNPV-Z and AnpeMNPV-L.By analyzing the number of SNPs in the three Antheraea pernyi nucleopolyhedrovirus,it was found that AnpeMNPV-L and AnpeNPV-Z share a closer genetic relationship than the others.We also analysis the distribution of SNP density throughout the genome,it was found that high-density SNP alway corresponds to the relatively lower G+C content in several region of genome.Caculating the Ka/Ks value of all AnpeNPV genes show that three genes have been undergone positive selection,which includes ODV-E56,ecdysteroid UDP-glucosyltransferase gene.It is notable that CTL-2(a copy of the gene conotoxin-like)contain premature stop condon that may result from the base mutations of AnpeNPV-H,indicating that AnpeNPV-H may reduce redundant genes to during adaptive evolution.