Studies On Polymorphism In The Morphologic,Ribosomal And Mitochondrial DNA Fragments Of Fasciola From DaLi And DeHong,Yunnan Province

[Objective]The objectives of the present study were to examine polymorphisms in the morphological,ribosomal and mitochondrial DNA(rDNA and mtDNA)fragments of Fasciola from DaLi and DeHong,Yunnan Province,as well as to investigate level of the species,genetic variation in Fasciola from DaLi and DeHong,Yunnan province.[Methods]1.Sample collection:Worms of Fasciola(n=40)were collected from the liver and gallbladder tube of cattle(n=3)and sheep(n=1)from two geographical locations of Yunnan province(DaLi,cattle(n=2),n=20;DeHong,cattle(n=1),sheep(n=1),n=20).2.Morphological study:Observe the shoulder of the Fasciola and the shape of the back end of the body by naked eye;measuring the body length and body width of Fasciola by a measuring ruler;observation of the characteristics of the shoulder of Fasciola and the genital cavity of Fasciola under microscope.Cone length,maxim-um diameter of oral sucker,maximum diameter of ventral sucker and distance between suckers were measured and recorded from printed images.3.Studies on polymorphism in the ribosomal and mitochondrial DNA:a.Extraction and identification of genomic DNA;b.Primer design and synthesis;c.The gene fragments of the internal transcribed spacer(ITS)and 28S rDNA regions of the ribosomal gene and the mitochondrial were amplified by PCR and sequenced;d.Program DNAStar,BioEdit and Mega5.1 were used for sequence analysis.[Results]1.The morphological analysis preliminarily suggested the existence of both F.hepatica and F.gigantica in Yunnan province.2.24 ITS sequences of 922bp and 16 ITS sequences of 902bp were obtained,28S rDNA sequences of 489bp were obtained from 40 worms.Sequence analysis display:the lengths of the complete ITS-1 sequences were 435bp with 5 variation positions,complete 5.8S sequences of 141 bp.While there was no nucleotide variation in the 5.8S rDNA among the 40 Fasciola samples.The lengths of the complete ITS-2 sequences were 346bp or 327bp with 8 or 5 variation positions.F.hepatica differed from F.gigantica at variation positions,and the "intermediate" type had both nucleotides of F.hepation and F.gigantica at these variation positions.40 specimens of Fasciola were identified as F.hepatica,F.gigantica and the " intermediate " Fasciola.3.The length of gene fragments of the mitochondrial nad4 was 470bp.Sequence analyses revealed that interspecific nucleotide difference in nad4 was significantly higher than intraspecific variation.The nad4 sequences of specimens representing the"intermediate" type were more similar to that of F.hepatica than they were to that of F.gigantica.The phylogenetic tree demonstrate:Fasciola from DaLi,Yunnan obviously assemble,have obvious branch with F.gigantica,but can't be constituted independent branch with the published sequences of the same mitochondrial nad4 were obtained from GenBank;Fasciola from DeHong,Yunnan obviously assemble,have obvious branch with F.hepatica,but can't be constituted independent branch with the published sequences of the same mitochondrial nad4 were obtained from GenBank.[Conclusions]1.morphological analysis of the preliminary suggested the existence of both F.hepatica and F.gigantica in DaLi and DeHong,Yunnan province.2.Taking the ribosomal ITS,28S rDNA as a genetic marker to identify the species of Fasciola from DaLi and DeHong,Yunnan province,the specimens of Fasciola were identified as Fhepatica,F.gigantica and "intermediate" Fasciola.3.Taking the mitochondrial nad4 as a genetic marker to study the genetic variation of Fasciola from DaLi and DeHong,Yunnan province,there were different degrees of genetic variation of Fasciola in DaLi and DeHong,Yunnan province.The intraspecific variation of Fasciola was not obvious,but the interspecific differentiation was more significant.