Linkage Mapping And Studies On Oocyte Maturation In Sea Cucumber Apostichopus Japonicus (Selenka)

Part I. Genetic linkage map constructiong of Apostichopus japonicusAmplified fragment length polymorphisms (AFLPs) and microsatellite markers were used for genotyping in sea cucumber Apostichopus japonicus. Sixty-two selected AFLP primer combinations produced 3572 fragments, of which 1133 (31.5%) were polymorphic in the mapping population. Each primer combination produced 18.3 polymorphic fragments on average. Among those 1133 polymorphic markers, 294 markers were from both female and male parents , 443 (52.8%) and 396(47.2%)markers were segregating through the female parent and the male parent respectively. Chi-square test indicated that 556 markers segregate in Mendelian ratio. 30 microsatellite loci screened were informative, 19 of them were heterozygous in the female and another 19 were heterozygous in the male parent.Mapmaker/EXP 3.0 software was used to process segregation analysis in the mapping population with 93 progenies. 358 markers were used in female segregation analysis, which involved 19 microsatellite markers. 199 AFLP markers and 11 microsatellite markers were anchored to the female linkage map. The female framework map is consisted of 25 linkage groups with the total length of 3148.3 cM. The max interval between markers is 37.5 cM and the average interval is 17.0 cM. The number of markers in each linkage group varies from 21 to 4. 332 markers were used in male segregation analysis. Among them 19 were microsatellite markers. 196 AFLP markers and 11 microsatellite markers were anchored in 23 linkage groups of the male framework map with the whole length of 3059.8c. Each of the linkage group contains from 4 to 18 markers and their average interval is 16.6 cM. The estimation of genome length of Apostichopus japonicus is 4053.7 cM for the female and 3816.3 cM for the male. The observe coverage was 84.0% for the female and 84.5% for the male.By using microsatillte makers heterozygous in both parents, three homologous pairs of linkage groups were identified. All markers were uniformly distributed without clustings. Some of the distorted markes were linked together.Molecular marker development and genetic linkage map construction provide the base for marker assisted selection, QTL mapping and comparative genome mapping. Part II. Research on oocyte maturation in Apostichopus japonicusThis research explored the methods of maturation induction of oocytes in vitro in sea cucumber Apostichopus japonicus. Using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and laser scanning confocal microscope, as well as technique of ultra-thin slicing and indirect immunofluorescent staining, we study the biological process of oocyte maturation in A. japonicus.Nerve extraction substance (NES) of sea star and dithiothreitol (DTT) has been reported to be effectively to induce oocyte maturation in sea cucumbers. In this study we tested their effects on oocyte maturation induction in sea cucumber A. japonicus. The results are as follows, oocyte of A. japonicus does not get mature spontaneously. Neither NES alone nor NES with follicle suspension can induce oocyte maturation. While DTT can prominently increase the percentages of germinal vesicle break down (GVBD). When the concentration of DTT is between10-1 mol/L and 10-3 mol/L, the effect of maturation induction is more sufficient. When the concentration is about 10-2 mol/L, the percentage of GVBD can reach up to more than 90%. Immatured oocyte of A. japonicus is arrested in prophaseâ… and is not fertilizable. After being treated with DTT, the germinal vesicle (GV) migrates and breaks down and the chromosomes are rearranged to the metaphase plate, followed by extrusion of the first and second polar body. The polar body extrusion process of induced maturation is similar to naturally fertilized eggs. Rising of fertilization membrane during induced maturation indicates insemination is not necessary to form the fertilization membrane. The oocyte will not become fertilizable until GVBD has happened and can still be fertilized after the second polar body extrusion. However, only those were fertilized before first polar body extrusion could process cleavage, and the embryos could not develop normally.Using light microscopy, electron microscope and confocal scanning microscope, we observed the process of DTT induced oocyte maturation in sea cucumber A. japonicus and analyzed the role of animal process of oocyte during ovulation and oocyte maturation. What's more, we studied the role of microtubules and microtubule organizing center (MTOC) upon GV migration. The oocyte of A. japonicus connects to the follicle via animal pole process. When placed in normal sea water, ovulation started spontaneously. The animal pole process punched in to the follicle and made a gap on it, and then the whole oocyte got out through this gap. Two forces may response to ovulation: the hydration of jelly space and contraction of follicle. Two MTOC are anchored beneath the animal pole process. Five types of microtubules were found in oocytes during maturation. After the onset of meiosis reinitiation, the GV stated to migrate along microtubules to the animal pole and attached to the site beneath oocyte membrane, that followed by GVBD. Oocytes treated with microtubule microtubule depolymerization agents before maturation induction failed to process GV migration. This result indicates microtubules are indispensable during GV migration. Afte GVBD, the two MTOCs begin to organize the meioses spindle. The animal pole process retracted back and the polar body extruded from the site of former animal pole process. This study fills up a gap in reproductive biology of A. japonicus.