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Alternative TitleStudies on Genetic Diversity of Transferrin and Related molecular Markers in Silver Crucian Carp, Carassius auratus gibelio
Thesis Advisor桂建芳
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline遗传学
Keyword银鲫 转铁蛋白 遗传多态性 有性重组 杂种起源 Dna多态性 选择
Other AbstractGynogenesis has been confirmed in the silver crucian carp (Carassins auratus gibelio); however, bisexuality has distinguished this population from other unisexual forms. A few clones have been successively identified from the gynogenetic crucian carp, which showed genetic diversity in karyotypes, histocompatibility, isozyme patterns, RAPD markers, etc. One of the main aims of this study is to elucidate molecular basis of genetic diversity of transferrin (TF) and other related molecular markers among silver crucian carp clones; with this issue as the beginning, I will subsequently discussed molecular fundamentals, reproductive modes, evolutionary origin and ecological adaptation of silver crucian carp. Therefore, four parts have been involved about this special fish in this dissertation: Firstly, TF polymorphism was found among clones of silver crucian carp and its molecular basis was elucidated by studying comparatively putative amino acid sequences of the TF variants. Altogether three TF alleles were identified for four clones of silver crucian carp and four ones were observed in an inbreeding population of colored crucian carp (Carasshis auratus colored variety). Clonal or Mendel's inheritance was shown for silver crucian carp and colored crucian carp respectively. Molecular weight variation of TF was also detected within two carp populations by SDS-PAGE analysis. Transferrin cDNA (77^-cDNA) was cloned from the two carps by RT-PCR and RACE-PCR and abundant sequence variations were observed among its variants. TF genotypes for six specimens of two carps were determined; they were A-C-B for clone D, A-C-D for clone A, A-E-?for clone F of silver crucian carp, and A_1A_1, A_1B_1, B_1B_1 for three individuals of colored crucian carp. Theoretical molecular weights (Mw) and isoelectric points (pI) of crucian carp TF variants were studied comparatively as well as their electrophoretic patterns, by which an empirical hypothesis was put forward to interpret their polymorphism on PAGE gel: mobility of a TF variant was negatively relevant to its Mw on SDS gel while negatively relevant to its pi on non-denatured gel. Relative mobility of TF variants has been predicted quite correctly by this hypothesis. Furthermore, PCR assays were practiced on TF gene of the two carps to supply an accessible approach to study its polymorphism on DNA level. Their TF genes were amplified by PCR and then analyzed by five restriction enzymes; tremendous polymorphism was observed among individuals on the digestion patterns. A definitely correspondent relationship was established among TF variants and their alleles in the colored crucian carp, by which Mendel's inheritance and co-dominant expression were also verified for its TF gene. At last, allele-specific PCR assays were practiced to detect TF alleles among silver crucian carp clones and expected results were obtained. Secondly, intraspecific reproductive modes of clone F of silver crucian carp were respectively studied by use of TF and isozyme markers. A female of clone F was mated with a male of clone D to produce FD line; while morphological differentiation and TF and isozyme polymorphism were observed in the progenies, protein phenotypes encoded by alleles from both parents were detected in some loci, which supplied a definite proof that sexual recombination had happened. Furthermore, extreme linkage disequilibrium was observed among protein loci and no homozygotic recombinant was detected for the loci, which suggested some characteristics of sexual recombination in silver crucian carp. Sexual recombination was intraspecific reproductive mode of silver crucian carp, which could remove mutation load and introduce new gynotypes to their genome. Alternative reproductive modes of sexual recombination and gynogenesis constructed biological foundation for generation, preservation and renaissance of clonal diversity in gynogenetic crucian carp. Thirdly, molecular phylogenies of TF gene and mitochondrial DNA control region (mt-CR) were respectively constructed among crucian carp populations to elucidate origin of Carassins auratus gibelio. Of the five TF alleles from gynogenetic C. a. gibelio, A was shared by three crucian carp clones and should have come from their common maternal ancestor. Since clone D and clone A shared two alleles A and C, and they also possessed the identical mt-CR sequence, probably they shared common ancestor of a diploid hybrid, thus C should have been introduced from paternal genome in the original hybridization, and alleles B of clone D and D of clone A could have been introduced in the second hybridization. With respect to clone F, E might have been caught from the paternal genome in the original hybridization. Alleles C and E of C. a. gibelio should have come from C. a. colored variety for their closely orthologous relationship with A\ and B\ respectively. Molecular phylogeny based on mt-CR data showed an independent maternal origin of C. a. gibelio; it also showed a relatively close phylogenetic relationship with C. a, colored variety. Of the three carp clones, clone F had diverged earliest while clone D and clone A diverged most recently. Generally, two hybridization events have happened in the generation of gynogenetic crucian carp: the first one occurred about 6,000 years ago, when maternal ancestor of C. a. gibelio hybridized with males of C. a. auratus (ancestor of C. a. colored variety) and a diploid hybrid was produced; the second one happened over 10,000 years ago, when the diploid hybrid was backcrossed with one of its parental population, by which the current triploid gynogenetic crucian carp emerged.Finally, DNA polymorphism of TF gene and mt-CR were studied among three populations of cruican carp. TF of crucian carp have exhibited polymorphism of a extremely high extent for its largest polymorphic parameters Pi and S compared with those data from other species. The abundant sequence variation, the large number and antiquity of alleles all suggested that selection could have driven the formation of TF polymorphism in crucian carp. However, two neutrality tests practiced on TF polymorphism, McDonald and Kreitman's test and HKA test, both refuted this deduction. Furthermore, no significant difference was observed between mean numbers of nucleotide substitutions per synonymous sites (Ks) and per nonsynonymous sites (Ka) among TF alleles. Probably the different regions of crucian carp TF had evolved at different rates and selection should be tested in different functional regions. Divergence of polymorphism of TF gene and mt-CR. were also studied among different crucian carp populations. Similar patterns of TF polymorphism were observed for crucian carp populations of different geographic distribution, while significant divergence also occurred between the continental population and that from Japan. By contrast, discontinuous ''break" was observed for mt-CR polymorphism among different crucian carp populations. Geographic isolation should have played the dominant role in divergence of DNA polymorphism for both sequences, however, bi-directional gene flow and the potential selection might have drive a common distribution of TF polymorphism among populations while the geographic "break" of mt-CR polymorphism might be relevant to its haploid and maternal inheritance.
Document Type学位论文
Recommended Citation
GB/T 7714
杨林. 银鲫转铁蛋白及其相关分子标记的遗传多样性研究[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,2002.
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