Heterosis Of Traits Related Growth And Sequence Analysis Of EST In Pearl Oyster, Pinctada Martensii

In order to improve the genetic quality of pearl oyster Pinctada martensii and develop new species, the author has designed a Pearl Oyster Breeding Scheme (POBS). The scheme aims to conduct a 2×2 diallel cross between two basic populations-- Indian cultured population and Chinese Sanya cultured population, and establish specialized paternal and maternal lines and breeding system through closed connective selection, in the hope of obtaining heterosis in growth rate (shell height and body weight) and marine pearl quality (color and nacre thickness) and developing better species. In order to carry out POBS, a series of researches has been conducted. First, the growth feature of P. martensii was investigated; then a complete 2×2 diallel cross between Indian cultured population and Chinese Sanya cultured population was conducted, hoping to obtain heterosis; after that, the application of heterosis to pearl oyster industry was explored, particularly the relationship between growth heterosis and genetic variation, potential for nucleus implantation and genetic effect, etc. Finally, EST analysis was performed in order to further molecular assisted selection and set up cDNA library.1. Growth features of cultured P. martensiiThe growth of P. martensii is characterized by fast initial growth of SH (Shell height) in the first year, followed by rapid increase of BW (Body weight) in the second year. The increase of SH was influenced by temperature, showing reduced growth rate in summer, fitting the extended Von Bertalanffy model: (?)(y: SH in mm, t: time in day). Growth rate of body weight increased steadily, except during typhoons or spawning, fitting the logistic model: (?)(y: BW in g, t: time in day). It is also established that in Hainan, most oysters were mature and suitable for pearl nucleus insertion after 16-20 months of farming; in order to meet the implantation season in April -June, it is more advisable to conduct hatchery operation in autumn than in spring.2. Genetic variation and heterosis of hybrids of two different geographic populations of pearl oysters P. martensiiA complete 2×2 diallel cross between Indian cultured population (II0) and Chinese Sanya cultured population (SS0)was conducted based on the following design: II₁: II0♀×II0♂IS₁: II0♀×SS0♂SI₁: SS0♀×II0♂SS₁: SS0♀×SS0♂The four groups of offspring were cultured under identical conditions in Li'an Lagoon, Linshui, Hainan Province.The results of the 2×2 complete diallel cross revealed that hybrid IS₁ and SI₁ showed heterosis in shell height (SH), shell length (SL), hinge length (HL), shell width (SW), and shell weight (SW). IS₁ had heterosis in shell width index (SWI), but not in body weight (BW) and shell weight index (SWTI), while IS₁ had heterosis in body weight and shell width index, but not in shell weight index. SI₁ exhibited significantly larger heterosis in SH, SL, HL, and BW than IS₁ (P<0.01), but significantly smaller in SW (P<0.01). Six selected microsatellite markers were used to analyze four lines of progeny. The results reveal that the FST was 0.375 and the average numbers of alleles were in the order of SI₁(6.17) > IS₁(6.00) > II₁(5.00) > SS₁(4.67); the average allelic richness per locus was SI₁(5.34) > IS₁(5.04) > II₁(4.47) > SS₁(4.55); the average expected heterozygosities (He) were IS₁(0.55) > SI₁(0.54) > SS₁(0.44) > II₁(0.42); and the observed heterozygosities (Ho) were SI₁(0.52) > IS₁(0.46) > SS₁(0.44) > II₁(0.42). The hybrids showed higher heterozygosities and genentic diversity than the purebreds, indicating that the cross increased heterozygosity and genetic diversity, and directly brought about heterosis.3. Growth heterosis and nucleus implantation potential of hybrids of two different geographic populations of pearl oysters P. martensiiThe von Bertalanffy was applied to the analysis of growth of four groups of progeny and the resulted revealed that the SH growth of IS₁ and SI₁ were faster than that of SS₁(p<0.01), slower than that of II₁(p<0.01). The fitted value y∞(theoretical maximum), and coefficient of growth k and growth indexφwere in the sequence of II₁>SI₁>IS₁>SS₁. Similarly, logistic model was adopted to analyze the BW of four groups and the results showed that IS₁ and SI₁ had faster growth of BW than SS(p<0.01), but slower than II(1p<0.01). Both the fitted value y∞(theoretical maximum), and growth indexφwere in the sequence of II₁>SI₁>IS₁>SS₁, but coefficient of growth k followed different order. The comparison of y∞,k andφwith their respective measured heterosis suggested that y∞can not only reveal growth rate, but also predict heterosis; therefore, y∞is an important growth index in genetic breeding of bivalves.Hybrids IS₁and SI₁ exhibited positive heterosis in SH and BW across all growth stages, but SI₁ had larger heterosis in SH and BW than IS₁. In addition, both hybrids manifested negative over-parent heterosis in comparison with II₁, but positive over-parent heterosis compared with SS₁. The larger heterosis of SI₁ than IS suggested that the parent species II had high quality, therefore demonstrated significant effect in changing the growth traits of local Sanya population.When shell height reaches 60mm, oysters are suitable for implanting nuclei of 6.5mm. 50% of II₁ group took only one year (12 months) to reach implantable size, but SI₁ and IS₁ needed 450 days (15 months) and 465 days (15.5 months) respectively. As II₁ have thin and fragile shell, thus unfit for nucleus implantation; by comparison, SI₁ are more suitable for pearl culture, because half are ready for implantation after only 15 months'cultivation. It can be concluded that the development of SI₁ as pearl producer will bring about shorter culture period and lower farming costs.4. Genetic effect and heterosis of growth-related traits of P. martensiiZhu Jun's additive-dominance model (AD model) and additive-dominance maternal paternal model (ADMP model) were applied to the analysis of hereditary law of growth-related traits of P. martensii.The seven traits: SH,HL,SW,BW,SWT,SWI and SWTI had larger ratio of additive variance than dominance variance to total variation, ranging from 30% to 88%, suggesting such traits can be improved through selection. The six traits, SH, HL, BW, SWT, SWI and SWTI manifested larger ratio of paternal variance than maternal variance to the total variation, with the ratio of maternal variance being zero. However, SW manifested larger ratio of maternal variance than paternal variance to the total variation, with the ratio of paternal variance being zero. Therefore paternal effects must be taken into consideration in improving the former six traits and mater effects must be considered in improving SW. The heritability of all traits reached significance level (p<0.01), narrow-sense heritability ranging from 0.15 to 0.69 and 0.30 to 0.88, broad-sense heritability from 0.33-0.90, suggesting both cross and selection are possible to achieve satisfactory results.In the hybrids between Indian cultured population and Sanya wild population, the six traits: SH, HL, SW, BW, SWT and SWI exhibited positive dominant effect, but SWTI showed negative one, reflecting that the hybrids of Indian cultured population and Sanya wild population can obtain heterosis in most traits.The prediction of average heterosis and over-parent heterosis of F1 and F2 suggests that (1) the average heterosis varied across growth stages, as at the late stage of growth only 3 traits (SH, SW and BW) of the seven traits manifested positive average heterosis and reached significance level while SWTI demonstrated negative average heterosis; (2) average heterosis of traits existed in F₁ and F₂ continuously; (3) The average over-parent heterosis being negative, therefore one of the parent group is too excellent to be excelled.5. The construction of cDNA library and analysis of expressed sequence tags of P. martensiiWe constructed cDNA libraries from adult tissues and sequenced 7128 ESTs. Clustering analysis identified 788 contigs (covering 5769 ESTs) and 1351 singletons, yielding a total of 2139 unique genes. Of these unique genes, only 935 had significant (E-value≤0.005) hits in GenBank and the remaining 1204 (56.3%) were novel. Most of the known genes are related to cellular structure, protein binding and metabolic processes. Putative host-defense genes (86) were identified including C-type lectin, ferritin, polyubiquitin, proteases, protease inhibitors, scavenger receptors, heat shock proteins and RAS oncogenes. The EST sequences developed in this study provide a valuable resource for future efforts on gene identification, marker development and studies on molecular mechanism of host-defense in pearl oysters.In summary, this research has confirmed the feasibility of Pearl Oyster Breeding Scheme through experiments and demonstrated the existence of heterosis in the hybrids between different populations of P. martensii., and advanced a series of theoretical guidance as well. First of all, growth-related traits were mainly subject to the control of additive effects and had high narrow-sense heritability, so such traits of parents can be improved through mass selection or family selection in the construction of specialized paternal and maternal lines through closed connective selection. Special emphasis should be placed on the six traits of SH, HL, SW, BW, SWT and SWI in the selection of paternal population and on SW in the selection of maternal population. Secondly, since the growth-related traits were also subject to the control of dominance effects and had high narrow-sense heritability, heterosis was able to be obtained through cross. The continuous existence of average heterosis in both F₁ and F₂ justified the efforts of genetic improvement by crossing Sanya wild population (SS)♀and Indian cultured population, which has laid a foundation for the advancement of molecular assisted selection.