Sequence And Phylogenetic Analysis Of COI And Nad1 Genes For Strongylidae Isolated From Equs

Strongylidae nematodes are the most important parasites in the equine large intestine,which seriously endanger the health of the hosts,making them sick or even die and leading tohuge loss to the animal husbandry.The use of a large amount of anthelmintic drugs has led to anthelmintic resistance in equine strongylids and high prevalence rate.Studies on the taxonomy and phylogeny of the parasitic nematodes in the equine are of great significance for the control of strongylosis.At present,foreign scholars have provided a wealth of datas for the study of Strongylidae,while there are few studies on Strongylidae in China.In this study,35 species of Strongylidae nematodes(collected from Xinxiang City,Henan Province)were identified by optical microscope,and then the mitochondrial COI and nad1 gene were amplified by PCR technology,exploring the phylogenetic relationshipof the Strongylidae.The amplified products were sequenced,obtaining 39 COI sequences and 32 nad1 sequences.By use of a variety of biological software,analysised the base composition and genetic distance of the target fragment.The maximum likelihood(ML)and Bayesian(BI)methods were used to construct phylogenetic tree based on COI and nad1 sequences,and a phylogenetic tree based on combined COI with nad1 was constructed.The conclusions are as follows:1.The length of the COI sequence was 393 bp,through multiple sequence alignment showed that the conserved sites,variable sites,parsim-informative sites and singleton sites accounting for 64.63%,35.37%,29.52% and 5.85%,respectively.The average content of A+T(67.3%)was significantly higher than that of G+C(32.7%),which showed obvious AT base preference.From the the point of amino acid codon,the average content of the third locus A+T was highest(78.6%),and the average value of the second locus A+T(63.1%)was slightly higher than that of the first locus(61.2%).Than the R value(transition/transversion)showed that the third codon position evolve fastest,the second codon sites without base substitution,had the slowest rate of evolution.The phylogenetic tree constructed based on COI sequences showed that individuals within a species are clustered into separate branches,the classification results between different genera is not satisfactory,but it still has a certain value.2.The length of the 32 nad1 sequences was all for 354 bp.Analysis the result of the multiple sequence alignment,conserved sites were 218,variation sites were 136,parsim-informative sites were 115 and singleton sites were 21.The average content of A,C,G and T was 25.4%,9.8%,21.7% and 43.1%,respectively.The average content of A+T(68.5%)was significantly higher than that of G+C(31.4%),showing the AT base bias.In terms of amino acid codons,the average A+T content highest(74.2%)was the third locus,while the difference of the average A+T content of the first loci(66.1%)and the second locus(65.1%)was small.Than the R value(transition/transversion)showed that the third codon position evolve fastest,the second codon sites had the slowest rate of evolution.In the phylogenetic tree,31 species of Strongyloid formed two groups,one group including Strongylus,Triodontophorus,Skrjabinodentus,caragandicus,Poteriostomum imparidentatum and Bidentostomum ivaschkini;other groups includeCylicostephanus,Cyathostomum,Coronocyclus Cylicocyclus,Petrovinema poculatum,Poteriostomum ratzii,Cylicodontophorus bicoronatusand,and Gyalocephatus capitatus.The Coronocyclus,Cyathostomum and Cyhcosiephanushad a near relationship,and C.elongatus,C.ultrajectinus had a far genetic relationship.The topology of phylogenetic tree constructed based on COI+nad1 sequences was similar to the former.3.Skrjabinodentuscaragandicus,obtained from the large intestines of horses in Henan province,was observed using light microscopy.ITS and COI sequences of S.caragandicus were amplifiedand sequenced using PCR techniques.The results showed thatS.caragandicus was medium-sized,walls of buccal capsule was S-shaped,much thicker anteriorly,the thickness of the front end was 10-12?m and the thickness of the posterior segment was 4-6?m.The external leaf crown consists of 8 long and broad elements,and the internal leaf crown consists of 16~18 short and broad elements.S.caragandicus with a well-developed esophagus funnel,the genital cone length of male was 337 ?m,and the gubernaculums length was 245 ?m;totallength of rDNA-ITSwas 853 bp,including 369 bp in ITS1,153 bp in 5.8S and 331 bp in ITS2,and the content of G+C of ITS1(47.2%)was significantly higher than that of the ITS2(40.7%);the length of COI was 393 bp,and A,T,G andCcontent were25.2%,42.7%,21.4%,10.7%,respectively.In thepresent study,S.caragandicuswas reported for the first time in Henan province.