Variation Detection Based On Next-generation Sequencing Of Type Chinese 1 Strains Of Toxoplasma Gondii With Different Virulence From China

Background Toxoplasma gondii is an intracellular protozoan that affects most species of endothermic animals including humans with a great infection rate. The vertical transmission of T. gondii causes abortion, constituting a serious threat to humans and leading to great losses in livestock production. Distinct from population structure of T. gondii in North America and Europe, Chinese 1(Toxo DB #9) is a dominant genotype prevalent in China. Among the isolates of Chinese 1, the Wh3 and Wh6 have different virulence and pathogenicity in mice. However, little has been known about their difference at the genomic level. Different genotype strains with disaffinity pathogenic mechanism and effector function is known as genotype/strain-associated pathogenesis. Intensive study the genome difference of the same genotype with significant phenotypic differences, it hasa great signifcance to explore the pathogenic mechanism of dominant genotype in China.Objective To explore the genome level differences of Chinese 1 genotype strains Wh3 and Wh6, and finding the critical effector may lead to of the phenotypic differences.Methods Collect the ascites from Wh3 and Wh6 strains infected mice, extract the genomic DNA after isolation and purification, then construct the genome sequencing library. Whole genome sequencing of Wh3 and Wh6 strains, bioinformatics analysis the differences of Wh3 and Wh6 when compare with type I GT1 strains, and specific retrieval the variation of Toxoplasma virulence related effector. using the q RT-PCR detect the expression level of those genes.Results We successfully sequenced the genome of Chinese 1 strains of Wh3 and Wh6. The average sequencing depths were 63.91 and 63.61 for Wh3 and Wh6, respectively. The variations of both isolates were identified in comparison with reference genome of type I GT1 strain. There were 505,645 and 505,856 SNP, 30,658 and 30,004 indels, 4,661 and 2,320 SVs, and 1,942 and 3,080 CNVs for Wh3 and Wh6, respectively. In target search variations of particular factors of T. gondii, the dense granule protein 3(GRA3) and rhoptry neck protein 3(RON3) were found to have 35 SNP, 2 indels and 89 SNP, 6 indels, respectively. GRA3 and RON3 were both found to have higher expression levels in less virulent Wh6 than in virulent Wh3. Both strains of type Chinese 1 share polymorphic GRA15 II and ROP16I/III with type I, II, and III strains.Conclusions Sequencing of the two strains revealed that genome structure of Chinese 1 and type I strains has considerable genomic variations. The GRA3 and RON3 may lead to the phenotypic differences, at the same time type Chinese 1 strains share the polymorphic GRA15 II and ROP16I/III with type I, II, and III strains.