Molecular Identification And Genetic Polymorphism Analysis Of Fasciola In Dali Prefecture, Yunnan Province

Objective:This study aims to identify the species of Fasciola flukes in Dali,Yunnan Province by molecular biological methods,analyze its genetic diversity,and verify"whether there is hybrid Fasciola flukes and the degree of heterozygous,so as to enrich the scientific basis for the classification of Fasciola in this region to some extent.To discuss the genetic diversity of inter-and intra-species and relationships between Fasciola hepatica and F.gigantica,,to look for the dominant species,so as to provide a theoretical basis for formulating the control strategies and improving the control measures of fascioliasis in Dali region,and to protect people’s health and reduce economic losses.In this study,multiple molecular markers were selected to facilitate the comparison with previous studies,to clarnfy the difference between the genetic polymorphism of Fasciola flukes in Dali and other regions,and to explore the application potential of different molecular markers.Methods:Nine bovine livers infected with Fasciola flukes were collected from a farmers’market in Xiaguan Town,Dali Prefecture,122 adult Fasciola flukes were isolated and tiheir eggs were incubated.Genomic DNA of 122 adult Fasciola flukes and their eggs were extracted,ribosomal DNA ITS+ sequences,as well as mitochondrial DNA NDA I and COX I sequences were chosen for amplification and sequencing,followed by alignment with GenBank data for homology analysis,and multiple PCR results of pepck gene were combined to determine the species of Fasciola flukes.BioEdit software was used to compare the sequencing results and further analyze the genetic variation of 122 adult Fasciola fluke and their first filial generations.Peak height ratio of alleles at specific heterozygous loci in ITS+ sequencing atlas,namely the ratio of heterozygous genes carrying F.hepatica and F.gigantica genes,was used to predict the heterozygous degree of different individuals.Fasciola flukes from Nanning,Guangxi Province were collected as control species for comparison.Results:ITS+ was 945/946bp in length and had 11 variable sites.Among the 122 Fasciola specimens,7(5.74%)were consistent with F.hepatica,and 115(94.26%)were Fg/Fh heterozygous flukes.In the sequence map of heterozygous flukes,the 11 variable sites showed double peaks,and the double peak ratios varied significantly among individuals,suggesting that the proportion of the genes of F.hepatica and F.gigantica is different,from Fg:Fh≥10:1 to Fg:Fh≤1:10.NAD I was 535bp in length and had 48 variable sites.The NAD I sequence of one specimen(0.82%)was identical to F.hepatica,while that of 121 specimens(99.18%)was highly homologous to F.gigantica.Among the 121 specimens,116 were completely identical,whereas the remaining five each had single base mutations.C.OX I was 438bp in length and had a total of 42 variable sites.Among the 122 specimens,the COX I sequence of one specimen(0.82%)was identical to that of F.hepatica,while that of 121 specimens(99.18%)were similar to F.gigantica.Of the 121 specimens,120 were completely identical,while the remaining one had one variable site.The multiplex PCR results for pepck showed that double bands for F.hepatica and F.gigantica could be amplified from all Dali Fasciola specimens,hence they were all heterozygous flukes.By combining the sequencing of ITS+,NAD I and COX I,and the multiplex PCR results for pepck,we found that all 122 specimens were Fg/Fh heterozygous Fasciola flukes.The sequence of mitochondria COX I and NAD I of the egg was identical with that of the adult.In ITS1 sequence,the egg and the adult were of the same type,but the degree of heterozygosity showed differentiation.Conclusion:The results of this experiment suggest that the Fasciola in Dali area are highly heterozygous.The identification results of NAD I and COX I molecular markers were similar.In 122 samples of Fasciola flukes,only one was F.hepatica and the rest were all.gigantica.Since mitochondria are inherited in maternal line,F.gigantica is the main maternal source of Dali Fasciola flukes.The ITS+ sequence divides the samples into F.hepatica(7)and heterozygous Fasciola flukes(115),but all the samples(7/122)shown as F.hepatica in ITS+ section were identified by pepck as heterozygotes.Therefore,in order to make the classification and identification results of Fasciola more accurate and reliable,multiple target genes should be selected for analysis.According to the differences of individual bases in different molecular markers,122 Fasciola flukes can be further divided into 8 gene subtype,with Fspl gene subtype accounting for 89.34%,which is the dominant subtype of Fasciola flukes in Dali area.