Establishment of genetic markers for breeding the white grouper ( Epinephelus aeneus ) Thesis

The white grouper (Epinephelus aeneus) is a protogynous hermaphrodite species found throughout the southern Mediterranean Sea and along the west coast of Africa to southern Angola. The species has a high market value, excellent taste, rapid growth rate, hardiness and relatively high disease resistance. Therefore, it is a promising candidate for intensive aquaculture and in 2010 an Epinephelus aeneus domestication project was launched by the department of fisheries and aquaculture of the Israeli ministry of agriculture. The mission of the project is to develop innovative technologies aimed at domestication, breeding, and industrial rearing of the white grouper. The major obstacles in this procedure are: Accurate identification of species by relying only on morphologic characteristic, monitoring of genetic variance reduction within the captive population, and understanding of Epinephelus aeneus reproduction strategy i.e. how many brooders within a group actually contribute genetic material to the offspring. The current research deals with these issues using several genomic tools. First a DNA identification method is applied based on sequence diversity in mitochondrial Cytochromec Oxidase C subunit 1 gene (COI) in order to identify the specimens used in the research. Phylogenetic DNAand protein-based trees of 36 different Epinephelus species were constructed to determine the correlation between phylogenetic distance and effective heterologous amplification of genetic markers in E. aeneus. The markers were used to determine the rate of polymorphism in the captive and wild populations. Finally, polymorphic microsatellite markers were used as parentity test to assign larvae (n=59) spawned by the captive broodstock to their parents. In addition, a larger population of the captive broodstocks' fingerlings was obtained in order to locate a population, using the parentity test set, which will form a segregating population for construction of the first generation linkage map. In this research a total of 656 published sequences of microsatellites in Epinephelus species were found, from which 233 were suitable for primers design. Heterologous markers were developed from eight different Epinephelus species (E. awoara, E. septemfasciatus, E. fuscoguttatus, E. itajara, E. guttatus, E. akaara, E. coiodes and E. lanceolatus). A cost effective two-step PCR method was applied for product amplification of each set of primers in which only two florescent primers were used for efficient detection and genotyping of all 233 PCR products. A total of 144 loci were found polymorphic and the yield of polymorphic microsatellites by species of origin ranged from 42 to 83%. The lowest yield was found for E. septemfasciatus (P = 0.001) which according to the Cytochrome c oxidase subunit I gene (COI) analysis was the most phylogeneticly distant from E. aeneus. However, all selected Epinephelus species were found suitable for development of heterologous markers. An average number of 6.2 alleles ranging from 2 to 14 per marker was found in the wild type population versus 3.7 from 2 to 7 per marker in the captive population (P= 1.85 e) suggesting a reduction in genetic variation among the captive broodstock population comparing to the wild. For parentage analysis of the offspring population a set of eight polymorphic microsatellites was developed. Among the twelve brooders two males and two females were found to contribute genetic material in various pairing combinations, whereas one pair was shown to be the parents of most of the progeny (69.2%) thus suggesting the reproduction mode of E. aeneus as polygamous. In light of the fact that only a small number of brooders contributed genetic material to the offspring and the detected decrease in allelic diversity among captive broodstock compared to the wild population of E. aeneus, the conclusion is that polymorphism level in the captive broodstock must be further monitored for potential loss of variance. Furthermore, future culture programs must enhance the captive stock genetic diversity by analysis of genetic markers. The parentity test is being used to establish a segregating population for linkage mapping of all available genetic markers. In the future additional markers can be developed using either next generation sequencing or heterologous amplification of known microsatellites in related species thus creating a white grouper linkage map that will be an infrastructure for domestication and breeding.