Selection Of DNA Barcoding Candidate Genes For Ciliated Protozoa

Biodiversity underpins and sustains mankind survivial and enviromentaldevelopment. It harbors consumptive, economical, social productive, aesthetic,cultural, genetic, medicinal and ecological values. And it provides the basis for theecosystems and the services they provide, upon which all lives on earth fundamenta llydepend. Beyond the tree traditional kingdoms of multicellular eukaryotic life(Animals, plants, fungi) making up almost all of the visible biosphere, there is onegroup including most of the eukaryotic microbes named protists also harborsabundant biodiversity and account for the catalogued species on Earth. However,there still are big gaps of microbes biodiverstiy study.As one of the flagship eukaryotic microbes, ciliated protozoa play important rolesin numbers of fields including: ecosystem function, disease in acuaculture, sewagetreatment processes, as model organism and bioindicators of environmental quality.During the long-term (at least700million years) evolution, ciliates becomes a largegroup with probably the greatest diversity of cell structure among all eukaryoticmicrobes. Therefore, this kind of unicellular organisms can provide valuable materialsand contribute to lots of basic biology majors, such as evolutionary biology, cellbiology, molecular biology and ecology, etc. And like any group of eukaryoticmicrobes, there are also big gaps of ciliates biodiversity study and this task is alwaysdaunting in biodiversity surveys. During my graduate study, we take advantage ofmolecular technology and focus on searching candidate genes for ciliated protozoaand to address the above issues. Main results for this study are as follows:1. Research on DNA barcoding candidate genes for four main group inOligohymenophorea and Spirotrichea:(1) Evaluation of Cox I based DNA barcoding technology in Paramecium: Paramcium,genus of ree-living and model organism that well studied ciliates in class Oligohymenophorea.The author evaluated the genetic variation of the Cox I gene in five Paramecium species andprovided148new Cox I sequence to GenBank. With genetic divergence comparison andnetwork haplotypes survey, the authors identified a total of90haplotypes and foundheterogeneity of Cox I gene in Paramecium. Furthermore, parsimony network andphylogenetic tree analyses revealed that intra-individual diversity had no effect in speciesidentification and only a minor effect on population structure. Our results suggest that the CoxI gene is a suitable marker for resolving inter-and intra-specific relationships of Parameciumspp. (2) Evaluation of efficiency nuclear gene based DNA barcoding among tintinnids: Inorder to better understand tintinnid ciliated protozoa, we sequenced and analyzed the SSUrDNA and ITS1-5.8S-ITS2regions of10species belonging to five genera in the orderTintinnida. The secondary structures of the ITS2region were compared among8closelyrelated genera, analyses based on SSU rDNA and ITS1-5.8S-ITS2regions sequence show thatdivergences within the family Tintinnidae are higher than that among other four families(Codonellidae, Ptychocylididae, Metacylididae and Codonellopsidae), suggesting thesubdivision of this family; species with agglutinated loricae are not clearly separated fromthat with hyaline ones; and, ITS2can be barcoding candidate gene for the related tintinids.(3) Association and evalutation of barcode gene among closely related Frontonia: Here,we associated four markers including rDNA (SSU, LSU,5.8S-ITS) and mitochondrial gene(Cox I) to evaluate the performance of them as candidate barcodes for the related Frontonia(including33samples and16species, provided192new sequence to GenBank) species,which are common and dominant in many aquatic biotopes. Molecular phylogenetics andgenetic measurements are applied in this work. Furthermore, morphological and ecologicalcharacters are also resurveyed and compared with the genetic results. The findings as:(1)high genetic divergence in cox1and ITS regions, which are more prominent than theirextremely similar morphological appearance;(2) the mitochondrial gene Cox I harbors moretraits than nuclear genes in species differentiations and may provide useful information onpopulation structures;(3) Cox I, ITS2and LSU-D2regions are moderately variable and canbe potentially used for related species identification,(4) there are three distinct subgroups ofthe genus Frontonia based on our phylogenetic analyses, and (5) molecular data can greatlyaid in species identification and estimation of genetic diversity(4) Searching suitable DNA barcoding gene for closely related species of Euplotes: In thiswork, we focus on the most species richness genus Euplotes (including60samples and22species, provided178new sequence to GenBank) and associated four markers includingrDNA (SSU, LSU,5.8S-ITS) and mitochondrial gene (Cox I) to evaluate the performance ofthem as candidate barcodes for these species. There was failure amplification of Cox I genefor most of the Euplotes species except E. vannus. By contrast, almost all of the nuclear geneswere amplified successfully (about99%). Secondary strucutre prediction of COI proteinsshows that there are different degrees of insertions in ciliates COI protein, and species of classSpirotrichea, including Euplotes, has an exaggerated insertion longger than that of classOligohymenophorea, thus making universal primer design much more difficult. Molecularphylogenetics and genetic measurements based on variable region of nuclear genedemonstrated that ITS2and LSU-D1/D2regions are more suitable for Euplotes barcodingstudy. Furthermore, we found that discords between genetic and morphological diversitiesexist in Euplotes species, which meaning that speciation and evolutionary process are verycomplex in this genus.2. Comparative mitochondrial genome in some protozoa and evaluation of Cox I basedDNA barcoding technology in ciliate(1) Mitochondrial genome of Acrasis kona (Excavata, Hererolobosea): In this work, theauthor participated in the Acrasis kona genome sequencing study, which based on the454 FLX+and Illumina GAIIx platform. We baited mitochondrial genome form the nextgeneration sequencing data using MITObim procedure and assembled them with MIRApackage. And finally, we got a size of51.5kb circular mitochondrial genome of Acrasis kona.The A+T content of these mitochondrial genome is high (83.3%). A. Kona mtDNA has40%annotated protein-coding genes missing and50%of tRNAs were similar with relatedNaegleria species. Homologs of DYW-type PPR and Thg1-like proteins implicated withRNA editing were also found in A. kona mitchondrial genome. Some Numts were alsochecked in the mtDNA, implying that lateral gene transfer had happened. The A. konamitochondrial genome study demostrates the high diversity of mtDNA evolutionaryprocesses.(2) Comparative mitochondrial genome analyses and phylogeny study based on COI ofdifferent eukaryotic lineages: There are ten mitochondrial genome available from the USNational centre for biotechnology information (NCBI, http://www.ncbi.nlm.nih.gov/), and thespecies of which are belong to Oligohymenophorea, Spirotrichea and Nassophrea classes.Comparative mitochondrial genome analyses demonstrate high genetic diversity amongdifferent ciliates lineages, and they have different gene contents and arrangement patterns. Tobetter understand ciliates Cox I gene’s variation and evolutionary process, we access to NCBIdatabase and download81COI protein amino acid sequences from species withmitochondrial genome and make phylogenetic analyses of them. The results show that ciliatehas very exotic insertion in its COI protein, which cause the seperated clade of ciliatedspecies in the phylogeny trees. This work demonstrates that ciliates mitochondrial genes havespecial evolutionary process and genetic mechanism. In one sense, mitochondrial Cox I geneof cliates is varied too much to apply as molecular marker for DNA barcoding study. Bycontrast, nuclear gene may be more suitable than Cox I gene in future biodiversity study.