| Recently, time saw a great advance in fish marine-culture industry, but fish breeding is still a bottleneck. Flounder, turbot and red sea bream are all the important marine-culture fishes in China. However, some issues such as genetic structure analysis of selected cultured populations, screening of anti-disease markers, genetic diversity evaluation of survival fish following sperm cryopreservation, and microsatellite marker development were seldom reported. In the present article, these concerned points were studied using AFLP, SSR, ISSR and RAPD technique. In detail, genetic variability was compared between common and two selective hatchery flounder populations using AFLP, SSR, ISSR and RAPD, and some candidate anti-disease markers were obtained in flounder using AFLP. In addition, genetic diversity was evaluated between fresh and frozen sperm fertilized turbot fry. Many microsatellite markers had been developed from red sea bream cDNA library. All these will be sure to improve the researches in fish genetic variation analysis, marker-assisted selection, economic traits gene mapping and genetic linkage map constructing. The main work and results are as follows:1. AFLP analysis was performed in order to evaluate genetic characteristics of one common population and two selective hatchery populations of flounder Paralichthys olivaceus. A group of 60 genotypes belonging to three populations was screened using ten different AFLP primer combinations. A greater number of total AFLP bands were observed from the common population than from the resistant or the susceptible populations. There were a total of 491 AFLP loci detected in common population, while they were 462 and 458 in susceptible and resistant populations, respectively. The total number of polymorphic bands was higher (P<0.05) in common population than in the other selected populations. The total polymorphic loci were 323 in common population (65.78%), 284 in susceptible population (61.47%), and 279 in resistant population (60.927%). Thepercentage of low frequency (0~0.2) polymorphic bands in the two selective hatchery populations was lower (PO.05) than that in the common population. The results confirmed that it was important to detect the genetic variability of the selective hatchery populations for the conservation of natural flounder resources.2. Twenty flounder individuals from susceptible and resistant population, respectively, were screened using 61 pair of AFLP primers. However no one marker found was completely dominant in one population and zero in the other population. Fortunately, there were 8 bands that showed significant gene frequency difference between susceptible and resistant populations. Considering the characteristics of quantitative trait loci, they might be the markers that linked to resistant gene, that is, candidate anti-disease markers, in which, 2 were in resistant population, 6 were in susceptible population. An efficient method for recovery of DNA fragment from denaturing polyacrylamide gels was developed to allow cloning of these markers. Then they were sequenced for application in fish selective breeding.3. Ten highly variable microsatellite loci were used to investigate one common population (30) and two selected hatchery populations (30, respectively) of flounder Paralichthys olivaceus. All of the ten microsatellite loci screened in this study showed marked polymorphism. A total of 76 different alleles were observed over all loci. The number of alleles per locus ranged from 3.67 to 8.67. The average of observed and expected heterozygosities ranged from 0.610 to 1.000. and from 0.536 to 0.821, respectively. A total of 14 unique alleles were found in common population, and 3 unique alleles each were found in susceptible and resistant populations. The effective number of alleles varied from 2.14 for Po35 to 5.60 for Po91. The number of genotypes ranged from 5.33 for Po56 to 12.33 for Pol. Compared with the common population, the two selected hatchery populations, susceptible and resistant, showed significant genetic changes such as fewer alleles per locus, a smaller number of low frequency alleles, a small number of unique alleles and a small number of genotypes, which indicated a reduction in genetic diversity.4. ISSR analysis was performed in order to evaluate one common population and two selected hatchery populations of flounder Paralichthys olivaceus. A total of45 individuals belonging to three populations were screened using 12 different ISSR primers. A total of 106 loci were produced in the three studied populations. 33.96%, 27.55% and 26.80% of these loci were polymorphic over all the genotypes tested in common, susceptible and resistant populations, respectively. The number of polymorphic loci detected by single primer combination ranged from six to 12. The average heterozygosities of common, susceptible and resistant populations were 0.1072, 0.0925 and 0.0913, respectively. Compared with the common population, the two selected hatchery populations, susceptible and resistant, showed certain genetic differences including a smaller number of total loci, a smaller number of total polymorphic loci (PO.05), a smaller number of genotypes (PO.05) and a smaller Shannon index, which indicated a reduction in genetic diversity. Intentional or accidental release of selected flounders into natural sea areas might result in disturbance of local gene pools and loss of genetic variability.5. RAPD markers were used to analyze one common population and two selected hatchery populations of flounder Paralichthys olivaceus. A total of 60 individuals belonging to three populations were screened using 12 different RAPD primers. A total of 71 loci were produced in the three studied populations. 43.48%, 42.42% and 42.19% of these loci were polymorphic over all the genotypes tested in common, susceptible and resistant populations, respectively. The average heterozygosities of common, susceptible and resistant populations were 0.1122, 0.1025 and 0.1113, respectively. Compared with the common population, the two selected hatchery populations, susceptible and resistant, showed some genetic differences including a smaller number of total loci, a smaller number of total polymorphic loci, a smaller number of genotypes and a smaller Shannon index, which showed a reduction in genetic diversity.6. In comparison with the common population, all were found a genetic diversity reduction in selected hatchery populations using four molecular markers, AFLP, SSR, ISSR and RAPD. Among the molecular markers to use random primers. AFLP, ISSR and RAPD, AFLP had produced the most bands, that is, covered the most loci in flounder genome, which indicated its high effectiveness. While microsatellite marker showed higher genetic discrimination ability than AFLP. ISSR and RAPD, if using suitable primers.7. In order to test if sperm cryopreservation affects genetic structure of turbot, SSR was used to analyze the genetic structure of turbot fry fertilized with fresh and frozen sperm. Turbot sperm was firstly diluted in TS-2 at a ratio of 1:1 and then cryopreseved by three-step method. Motility score of frozen-thawed sperm was 65% and fresh sperm was 85%. Turbot fry fertilized with frozen and fresh sperm. 15 used for analysis each, were from same parents and it decreased the effect of parents' genetic type on the results. 30 alleles and 25 genotypes were acquired from 15 turbot fry fertilized with frozen sperm at 10 microsatellites DNA loci, which was the same as results from 15 turbot fry fertilized with fresh sperm. The heterozygosity was calculated on the basis of alleles frequencies of each microsatellite locus. The total heterozygosity of turbot fry fertilized with fresh sperm was 0.5747, while that of turbot fry fertilized with frozen sperm was 0.5714. In 30 alleles, only 7 allele frequencies varied significantly.8. Red sea bream cDNA library were constructed. By sequencing randomly, 2010 ESTs were obtained. With the bio-soft SSRHunter 1.3, 103 microsatellite repeat sequences were found in the sequences. In the 103 repeat sequences, the number of the dinucleotide repeats were 73, and most (70.67%) among all of the repeat sequences; the second were the trinucleotide repeats, 22 (21.36%); the third was the tetranucleotide repeats, 7 (6.80%); the fourth was the petranucleotide repeats, 1(0.97%). No hexanucleotide or longer sequence repeats were found. In dinucleotide repeat, the numbers of AG repeats were 33, the most, accounting for 32.04%; and the second and third were AC and AT repeats, 30 (29.13%) and 10 (9.71%) respectively; no GC repeat was found. Eight sorts of repeat sequences that respectively composed of the motif AAT, AAG, ACG, AGG, AGC, ACT, GCC and ATC, were found in the trinucleotide repeats, in which the numbers of AAT repeats were 6 (5.83%), the most; the second, AGC were 4 (3.88%); the following were AAG, ACG and AGG, 3 (2.91%); ATC, GCC and ACT, the last were all 1 (0.97%). ACCA, CACT, CTTT, GTAT, CTGT, GCCA, AAAT, 1 (0.97 %), respectively, were the tetranucleotide found in the 103 microsatellite-containing ESTs. AATCT (1, 0.97%) was the only hexanucleotide.9. Microsatellite markers had been developed from a cDNA library of red sea bream, Chrysophrys major. 28 microsatellites were selected for designing microsatellite primers, of which 11 gave working primer pairs. The number of alleles per locusranged from 2 (CM000278 and CM001034) to 8 (CM001742), and the observed and expected heterozygosities ranged from 0.33 to 1.00, and from 0.38 to 0.83. respectively. Only 5 of 11 loci conformed to Hardy-Weinberg equilibrium (HWE). The remaining six loci all significantly deviated from HWE, one of them (CM002640) showed a large heterozygote deficiency and five of them show significant heterozygote excess. Out of 55 possible pairwise comparisons between the 11 loci applied to red sea bream, none showed significant linkage disequilibrium. Five additional fish species assessed for cross-species amplification revealed between one and six positive amplifications and between 0 and 6 polymorphic loci.
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