| Blunt snout bream (Megalobrama amblycephala) or Wuchang Fish, belongs to the family of Cyprinidae, which is an herbivorous freshwater fish species native to China. It was originally endemic to certain large and medium-sized lakes of middle reach of Yangtze River, such as Liangzi lake (LZ) and Yuni lake (YN) in Hubei province and Poyang lake (PY) in Jiangxi province.Because of its special cultural elements, high resistance to diseases, high rate of meat and rich nutition, M. amblycephala has been widely favored and recognized as a main aquaculture species in the freshwater polyculture system since1960s in China. Due to its successful artificial propagation and high economic value, M. amblycephala aquaculture industry has greatly developed in the past decades and its total output had reached652,21tons in2010(CAFS,2010). As a consequence of fast domestication, over-fishing, however, germplasm resource of this fish is under threat of recession and mixture due to its artificial breeding. Nowadays, the cultured population of M. amblycephala is gradually exhibiting early sexual maturity, low growth rate and disease susceptibility. Therefore, applying genomics tools in the selection of broodstock has the potential to enhance the productivity and value of commercial production for this species.Right now, the molecular genetic basis of M. amblycephala is very weak and genetic breeding theory is not mature, serious influencing genetic breeding program of M. amblycephala. In order to carry out advanced breeding programmes more effectively as soon as possible, and to breed better varieties, transcriptome of M. amblycephala using next genration sequencing (NGS) technique was first sequenced. And then, we developed many microsatellite markers based on these EST sequences. We used eight microsatellite markers to build a parentage assignment technology platform, and based on this technology, detailed study was conducted on quantitative genetics of M. amblycephala F1generation. In this thesis, we combined with the theoties of quantitative genetics, cross breeding and molecular-assited selection (MAS), and used the methods of family selection, mass selection, BLUP breeding and progeny testing to carry out a breeding programme of M. amblycephala. This research lays the foundation for gaining good varieties of M. amblycephal in future. Our results and conclusions are as follows: 1. De novo assembly and characterization of M. amblycephala using454pyosequencingA total of1,409,706high quality reads (total length577Mbp) were generated from the normalized cDNA of pooled M. amblycephala individuals using454pyosequencing. These sequences were assembled into26,802contigs and73,675singletons, with7.6×sequencing depth. The average length of contigs was730bp, and more than70%of contigs were longer than500bp. After BLAST searches against the Nr and UniProt databases with an arbitrary expectation value of E-10, over40,000unigenes were functionally annotated and classified. Of which, a substantial number of candidate genes putatively involved in growth, development, reproduction and immune were identified, and were considered as an invaluable genetic resource for gene functional analysis and breeding program. A comparative genomics approach revealed a substantial proportion of genes expressed in M. amblycephala tanscriptome to be shared across the genomes of zebrafish, medaka, fugu, stickleback, tetraodon, chicken, mouse, and human, and also identified a lot of potentially novel M. amblycephala genes. A total of4,952SSRs were identified and93polymorphic loci have been characterized. A large number of SNPs (25,697) and indels (23,287) were identified based on specific filter criteria in M. amblycephala.2. Development of SSR markers from EST sequencesIt is a low cost and simple to development of SSR markers from EST sequences. In this study,300SSR loci were randomly selected for primer design, of these,146loci (48.7%) were successfully amplified and93loci (31%) were polymorphic across panels of40individuals from LZ population. Number of alleles (NA) is from2to18, with an average of5.11. The observed heterozygosity (Ho) and the expected heterozygosity (HE) ranged from0.25to1and0.25to0.86, respectively. In order to analysis population genetic diversity or genetic structure more efficient and more accurately, six multiple PCR reaction systems were constructed with20polymorphic microsatellite markers to analysis the genetic diversity of PY population. The results indicated the genetic diversity of PY population is a little low, and most loci were significantly deviated from Hardy-Weinberg equilibrium, indicating the M. amblycephal wild population suffered serious artificial interference. 3. Construction of a parentage assignment technology for M. amblycephalaIn this study, we used eight highly polymorphic microsatellite markers to assign parentage to offspring under different scenarios both simulation and real. Simulations based on allele frequency data from the population of M. amblycephal demonstrated that4loci were required to assign91.3%and6loci to assign99.6%of progeny. In a real-life commercial breeding system, using8SSR loci with NA of10, the assignment success of progeny to their true parental was98%with known parental and filial information and96.33%in mixed families groups.4. The growth performance of F1generation and rapid growth families screening317full-sub F1families were constructed by a complete diallel crossing of three strains (Liangzi-LZ, Poyang-PY and Yuni-YN). All the F1progeny were fed in a communal pond from newborn until to when harvested. Based on microsatellite parentage assignment techniques,708(94.53%) of749offspring could be assigned directly to a single parental pair. Finally,25families with body weight (?0400g were screened, which could be as core breeding families in M. amblycephal selection program. In addition, based on these body length and weight growth fastest and growth slowest families, the body length and weight relationship was constructed with correlation coeffient R2>0.5. The a values of two equations were similar, indicating these families were in stable growth environment; The b values were both<3, implying these families were in allometric growth stage, which is in accordance with the normal growth rule of M. amblycephal.5. Heterosis and combination ability analysis in a diallel cross of three populations of M. amblycephalaIn this study, a complete diallel crossing of three strains (LZ, PY and YN) was used to evaluate the combining ability and heterosis effects of intraspecific crossbreeding on its growth performance. The offspring produced between the three strains were reared in a communal pond for20months and9microsatellites were used to pedigree. Significant differences were observed in growth among different mating combinations, indicating combining ability of body weight (BW) from each strain was significantly different. General combining ability (GCA) of BW from sire is much higher than dam. Both the GCA for dam and sire of LZ was the largest and that of YN was poorest. Special combining ability of YN(?)×PY(?) was the largest and YN(?)×PY(?) showed significantly positive heterosis effects on F1growth (P<0.05). A significant and positive linear correlation existed between the growth of progeny and the level of genetic variation in the parental generation.6. Estimation genetic parameter and breeding value for growth traits of M. amblycephalaThe genetic parameters estimates for important economic traits are needed for its selective breeding. The aim of the current study was to estimate the heritabilities for its growth-related traits and explore the genetic and phenotypic correlations among the traits using microsatellite-based pedigree approach. Offspring from a mass-spawning of92broodstocks (42sires and50dams) were reared in a communal pond and nine microsatellites were used to identify the parents of each sampled offspring. Data was analyzed by the method of restricted maximum likelihood (REML) using animal model and the results showed the heritabilities of body weight, body length, total length and body height were0.65,0.53,0.53and0.50, respectively. High genetic correlations were found among these four traits. According to these results, selection for growth seems to be feasible in M. amblycephala and the other growth traits will be heightened accordingly with the selection based on body length. In addition, breeding value for growth-related traits of20-month M. amblycephala was estimated by the BLUP method. The BLUP breeding value for single trait (body weight or body length) was similar with combined breeding value, which took full into account the body type. The results shown most of the parents with high breeding value were from Liangzi strain and Poyang strain.7. Assessment of parental contributions to progenies in M. amblycephala and selection of parents with good traitsThe parentage assignment base on microsatellite markers was used to assess the parental contributions to fast-and slow-growing progeny group.95.32%individuals could be unambiguously assigned to their putative parents. The FST value was0.024(p<0.05), implying significant genetic differences between the two progeny groups. A globally significantly unequal contribution of breeders to fast-and slow-growth progeny was found in this experiment.15breeders (6females and9males) contributed significantly more to the fast-growth progeny, while11(4females and7males) contributed significantly more to the slow-growth progeny. In addition, we estimated the BLUP breeding value for body weight of parents, and found that the breeding value of the selected parents were all much high. The result indicated it is reliable to select excellent parents by estimating parents’ contribution to progeny. These excellent breeders could be used to establish good families as core selection group and could be directly used in production mass fry. The findings are helpful to perfect the selective breeding program of M. amblycephala and also gave some advices for breeding selection in other fish species. |
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