Studies On Polymorphisms In Ribosomal ITS And Mitochondrial DNA Sequences Of Chabertia Spp

Chabertia belongs to Strongyloidea of Strongylata which lives in intestines of ruminants,such as cattle and sheep, with a global distribution. Chabertia ovina and Chabertia erschowiare two common Chabertia species with taxonomic controversy. The present study involvedthree parts: to examine DNA polymorphisms in internal transcribed spacers (ITS) of ribosomalDNA and in mitochondrial genes, and to determine the complete mitochondrial genomesequences of C. ovina and C. erschowi.In the first part, we examined sequence variability of ITS rDNA among C. ovina isolatesfrom goat in Shaanxi province and C. erschowi isolates from yak in Qinghai province. The ITSrDNA was amplified by PCR from individual Chabertia samples, and then the PCR productswere cloned, sequenced and analyzed. Among the6C. ovina samples, the variation was0~1.1%for ITS-1sequences and0~1.7%for ITS-2sequences, while the the variation in ITS-1andITS-2sequences were0~1.9%and0~1.7%among9C. erschowi samples. However, theinter-specific sequence differences were significantly higher, being6.3%~8.2%for ITS-1and10.4%~13.6%for ITS-2, respectively. The results showed that C. ovina and C. erschowi maybe distinct species.In the second part, four mitochondrial genes of C. ovina and C. erschowi, namely partialcytochrome b (pcytb), partial NADH dehydrogenase subunit4(pnad4), complete NADHdehydrogenase subunit2(nad2) and complete small subunit ribosomal RNA (rrnS), wereamplified by PCR, cloned and sequenced, then the DNA polymorphism was studied. Thelengths of pcytb, pnad4, nad2and rrnS for C. ovina were659bp,914bp,840bp,696bp,respectively. The lengths of these four genes were659bp,810bp,840bp,696bp for C.erschowi, respectively. Results of sequence analyses between C. ovina and C. erschowiindicated that interspecific variations in four mtDNA genes were high, namely15%~17.3%forpcytb,16.4%~17.7%for pnad4,18.1%~19.4%for nad2,6.9%~7.5%for rrnS, respectively.Compared with C. ovina Australia isolate, C. ovina Shannxi isolates had lower differences inthese four mitochondrial genes, being4.9%~5.6%for pcytb,5.1%~5.6%for pnad4,3.7%~4.5%for nad2,0.9%~1.3%for rrnS, respectively, while C. erschowi Qinghai isolates had relativelyhigher differences, being16.1%~16.8%for pcytb,16.9%~17.3%for pnad4,19.2%~19.9%fornad2,6.9%~7.3%for rrnS, respectively. These results showed that pcytb, pnad4, nad2andrrnS provide novel genetic markers for molecular taxonomy of Chabertia parasites, and supported the hypothesis that C. ovina and C. erschowi may be separate species.In the third part, the complete mitochondrial genome sequences of C. ovina and C.erschowi were amplified, cloned, sequenced and then analysed. The results showed thatcomplete mitochondrial genome of C. ovina and C. erschowi were13717bp and13705bp insize, respectively. Both genomes are circular, and totally compose of37genes, which include12genes for proteins,2genes for rRNA and22genes for tRNA. Phylogenetic relationship with14nematodes was reconstructed by Bayesian inference based on concatenated amino acidsequences of12protein-coding genes. The results further indicated that C. ovina and C.erschowi may represent distinct species.In summary, this study examined sequence variation in ITS rDNA and four mitochondrialgenes, and determined the complete mitochondrial genomes of C. ovina and C. erschowi. Takentogether, these results indicated that C. ovina and C. erschowi represent different species. Theseresults not only provided new data of mitochondrial sequences for the studies of nematodes, butalso have important implications for molecular taxonomy and population genetic structures ofnematodes.