Study On Induction Of Allogynogenetic Tetraploid And Alloteraploid In Scallop And Its Cytogenetic Analysis

Allogynogenetic tetraploid of scallop Chlamys farreri was induced by UV irradiated heterogenous sperms of the Pacific oyster Crassostrea gigas, and artificial allotetraploid of scallop Chlamys farreri was induced using sperm from Patinopecten yessoensis and eggs from Chlamys farreri in this paper. The cytogenetic analysis techniques, such as chromosome contribution, squashing method, paraffin section, fluorescent staining, and GISH were applied to study the tetraploid induction process and cytogenetic analysis. New ways for induction of tetraploid scallop were provided in this study. The main content and results were as follows:Part One: Induction of allogynogenetic tetraploids in Chlamys farreri1.1. Genetic inactivation of the Pacific oyster spermAllogynogenetic haploid of scallop Chlamys farreri was induced by genetic inactivated sperm of the Pacific oyster Crassostrea gigas. Heterogenous sperms were ultraviolet-irradiated at an intensity of 1500μw/cm2. The results showed that UV irradiation of the sperm for 60s at intensity of 1500uw/cm2 was the appropriate dose to achieve haploid gynogenesis on the basis of the results of fertilization rate, percentage of haploid, and survival rate of early embryos; The fertilization rate decreased with increasing irradiation time; The survival rate of early embryos suggested the presence of a"Hertiwig effect"in the gynogenesis of Chlamys farreri.1.2. Induction of allogynogenetic tetraploid in scallop Chlamys farreriAllogynogenetic tetraploid of scallop Chlamys farreri was induced by UV irradiated heterogenous sperms of the Pacific oyster Crassostrea gigas. The induction was attempted by blocking the release of the first and the second polar bodies in the fertilized eggs with 6-DMAP (50mg/L, 35min). The Pacific oyster sperm were ultraviolet irradiated for 60s at an intensity of 1500μw/cm2. The ploidy of resultant trochophors was determined by chromosome counting. Trochophors from the treatment group was consisted of 6.25% tetraploids, 8.33% haploids, 13.54% diploids, 5.21% triploids and 66.67% aneuploids. The results showed that allogynogenetic tetraploid could be obtained with the method of gynogenesis in this paper. But more work need to be done to raise the percentage of tetraploid.1.3. Cytological observations of nuclear behaviorThe cytological process of the fertilization and early embryonic development of allogynogenetic tetraploid were observed under fluorescent microscope with DAPI stained. The process was also observed under an optical microscope with the method of paraffin section. The results of cytological observation revealed that UV inactivated sperm of the Pacific oyster could be inseminated with scallop eggs and activate the fertilization process even though the development was delayed. The nucleus of ultraviolet irradiated sperm expanded slightly after penetrating into eggs of Chlamys farreri. At metophase of the first cleavage, the male pronucleus became a dense chromatin body (DCB), which did not participate in the karyokinesis and located between the two maternal chromosomes. At completion of the first cleavage, DCB was observed either in the region of the first cleavage furrow or in one of the two blastomeres. During the second cleavage, the experience of DCB was fundamentally identical to the first cleavage. Treatment with 6-DMAP effectively blocked the release of the first and the second polar bodies, and resulted in tetraploidy female pronucleus. Chromosome investigation documented that allogynogenetic tetraploid did be obtained in fact. In addition, the embryos with varied ploidy, irregular division of nuclear materials, and chromosome pluripolar segregation were observed and discussed.Part Two: Induction of allotetraploid in cross-fertilized eggs of Chlamys farreri♀×Patinopecten yessoensis♂2.1. Induction of allotetraploid and optimization of induction parameter In order to find out the appropriate duration time of 6-DMAP, ploidy status of induction offspring was analyzed by chromosome distributions and flow cytometry. The results showed that the optimal duration time of 6-DMAP was to treat the fertilized eggs with 50 mg/L 6-DMAP for 15min based on the integrated evaluation of the results of fertilization rate, survival rate of D-larvae, development status of larvae, and percentage of tetraploid. The results of flow cytometry showed that 17.3% tetraploid was obtained. 6.84% tetraploid was gotten with the method of chromosome counting.2.2. Cytological observation of chromosome segregation patternChromosome segregation in fertilized eggs from Chlamys farreri♀×Patinopecten yessoensis♂following inhibition of the first polar body with 6-DMAP, was studied with hematoxylin staining techniques. Cytological observation indicated that the inhibition of PB1 with 6-DMAP changed the normal chromosome segregation pattern. Multipolar segregation patterns were detected in the second meiosis. Three typical types of segregation were observed, namely,"united bipolar segregation"(75.50%),"separated bipolar segregation"(6.2%), and"triploar segregation"(7.39%). There were 10.91% of treated eggs that could not be classified. The integrated analysis of segregated types and results of trochophores ploidy indicated that"separated bipolar segregation"might lead to the formation of tetraploids."United bipolar segregation","triploar segregation", and segregation patterns which could not be classified would mainly produce aneuploids. The possible mechanism for the various types of chromosome segregation was discussed.2.3. Cytological observation of nuclear behavior in early embryosThe nucleus behavior in fertilized eggs from Chlamys farreri♀×Patinopecten yessoensis♂following inhibition of the first polar body with 6-DMAP were investigated under fluorescent microscope with DAPI stained. The developmental process of zygote was complicated and was classified three types which were two polar body releasing (30.9%), one polar body releasing (54.35%) and none polar boy releasing (14.75%). The release status of polar bodies will result in diploid, triploid, tetraploid, pentaploid, and aneuploid. In addition, the embryos with irregular division of nuclear materials and gynogenesis were observed.2.4. Spindle and chromosome behaviors in early embryosSuitable egg fixation and solution of fixation were selected and modified by a series of advance experiments. The results showed that the proper egg fixation method for microtubule skeletal immuno-fluorescent micro-observation in Chlamys farreri was to be fixed with 4% paraformaldehyde fixation solution, then be stored in 0.1M PBS(pH7.4) at 4℃. Spindle and chromosome behaviors were observed by fluorescent staining method using FITC-anti-α-tubulin and propidium iodide (PI) respectively. Great changes of spindle happened during the first and second cleavages in fertilized eggs. Several types of meiotic apparatuses and corresponding chromosome segregations including"tripolar segregation"and"double bipolar segregation"were observed in accordance with the results of DAPI stained. The results of nuclear behaviors and the phenomenon of gynogenesis were observed which were in accordance with the results of DAPI stained, too.2.5. Cytogenetic analysisThe genomic constitution and its variation in larvae at six periods from induced offspring of allotetraploid were studied by genome in situ hybridization and traditional chromosome preparation techniques. The results showed that 5.192% allotetraploids and large proportion of aneuploids and allotriploids were obtained. The unequal and ruleless chromosome contributions were observed except the allodiploid by GISH technique. Allodiploid inherited one set of chromosome from each side of the parents. But allohaploid, allotriploid, allotetraploid, allopentaploid, and alloaneuploid didn't inherit euploidy sets of chromosomes from the parents. The majority of chromosomes of these offspring were inherited from their maternal parent except the allodiploid. These results may suggest that the two sets of chromosomes from the two scallop species have great compatibility in the zygotes. There were also some incomplete-inherited individuals in the offspring. In addition, the reasons for low induction rate and poor survival of allotetraploid production and the implications and potential applications for aneuploid were discussed.