The development and application of DNA markers for the American lobster, Homarus americanus

The American lobster (Homarus americanus) fishery is a multi-million dollar industry, along the north Atlantic coast of North America. Sustainable management practices require information on the nature and degree of genetic diversity that exists within and among lobster populations. The objectives of this study were twofold: first, to identify and characterize candidate molecular markers; and second, to test populations off Prince Edward Island, Canada for homogeneity using genetic markers. Direct (diploid) sequencing of PCR amplicons was used to screen for allelic variation within an intron of the cytoplasmic gelsolin (CyG) gene. It was observed that diploid sequencing can be useful as a screening tool for allelic variation, however additional complimentary molecular analyses are required to accurately characterize length polymorphisms (indels) such as the microsatellites identified within an intron of CyG. Candidate CyG alleles identified by diploid sequencing were characterized further by sequencing of cloned amplicons containing one of the microsatellite regions and generated using low fidelity (Taq) and high fidelity (Pfu) DNA polymerases. Among eight individuals analyzed, seven alleles were identified from a total of 45 Taq-generated clones. Among 54 Pfu-generated clones only three alleles were identified. Discrepancies between the results were attributed to nucleotide incorporation errors that occurred either during the PCR, cloning or sequencing process. The genetic analysis of population variability is dependent on the quality of the data produced therefore all measures to minimize nucleotide errors and an independent verification method should be performed. A gene-associated microsatellite locus identified within the 3' untranslated region (UTR) of the crustacean hyperglycemic hormone B gene (CHH-B) was used to examine the genetic variation and population structure of lobsters from six geographic locations off PEI. There were extensive polymorphisms within the locations and allelic richness values standardized for N=18 indicated there were differences in the number of alleles between populations. All populations were in Hardy-Weinberg equilibrium (HWE) and no significant population substructure occurred among the locations (FST 0.05), suggesting mixing is occurring among the locations. Sequences for two genes involved in arthropod immune function were isolated from H. americanus. A 2672 by full length cDNA encoding prophenoloxidase (proPO) was obtained from haemocytes by reverse transcription polymerase chain reaction. The deduced polypeptide sequence of 673 amino acids showed highest similarity with other known decapod proPOs. A 3902 by genomic sequence for a Toll-like gene (Ha-Toll) was identified using nested inverse PCR. A 196 amino acid sequence was predicted and shows similarity to several insect Tolls as well as a recently identified chelicerate Toll. This novel sequence data represents two genes intimately involved in immune function and creates a platform for future studies in population health and evolutionary analyses in homarid species. The application of genetic markers provides information concerning a species' genomic diversity. Three genetic markers as well as two novel gene sequences for H. americanus were identified in this thesis. A preliminary study with one microsatellite marker was conducted to examine the genetic variability between north and south shore lobster populations off Prince Edward Island. Although the results suggested a panmictic population, additional studies using a larger number of genetic markers and greater sample sizes should follow to further test the hypothesis of genetic homogeneity and strengthen the conclusions made concerning lobster stocks.