Workpackages

Stock identification of horse mackerel is still doubtful and largely based on managerial criteria rather than on a scientific background. With the aim to identify doubtless the boundaries of the various stocks on genetic bases, the genetic structure of horse mackerel populations will be investigated throughout the whole range of the species. Different molecular approaches will be used, in order to magnify their resolution power and to validate the results obtained with each single technique: multilocus allozyme electrophoresis (MAE), microsatellite DNA (msDNA), mitochondrial DNA sequencing (mtDNA), random amplified polymorphic DNA (RAPDs) and single-strand conformation polymorphism (SSCP).

2.1 Multilocus allozyme electrophoresis (MAE)
The MAE will provide basic data to evaluate the degree of genetic structuring of populations and the genetic diversity of horse mackerel samples. A statistically significant number of specimens (at least 100) will be analysed from each of the 20 selected sites by means of standard horizontal starch-gel electrophoresis. Allozyme electrophoresis is a methodology able to provide a large number of genetic markers (40-50) and has been proved powerful in providing reliable results in stock assessment of pelagic fish. With respect to many other genetic approaches multilocus electrophoresis is easier to set up and quicker in providing reliable results easily comparable with the largest bulk of the data reported in literature. The expected results will provide data on the horse mackerel stock boundaries (genetic diversity partitioning); on the degree of isolation of each detected unit respect to the others (gene flow indirect estimations); on the genetic relationships among the samples.

2.2.- Mitochondrial DNA (mtDNA)
The mtDNA genome is an excellent system for population analysis because of its easy isolation, absence of recombinations, taxonomic homology and its mainly maternally inheritance through the oocite. Specific gene segments can be amplified via PCR by using a pair of primers in quantities sufficient for direct sequencing. This technique can detect single base pair changes and, therefore, provides a large number of characters for comparative purposes. The ND genes have been examined in a number of species as they are reported to exhibit variability on the population level. The control region has the highest substitution rate of all mitochondrial genes that makes it very suitable in populational studies. A number of significant samples will be collected from each area (at least 100) and the tissue samples analyzed. Finally, appropriate statistical tools will be applied to identify populational characteristics.

2.3.- Microsatellite DNA (msDNA)
The use of highly variable microsatellite DNA markers in marine species can uncover subtle population genetic structuring, even in cases where other types of markers failed to do so. Individual microsatellite loci can be studied either by developing primers specific to unique flanking domains of individual microsatellite loci , allowing amplification and description of individual alleles, or by cloning the entire VNTR or one or both domains of unique flanking msDNA and using this to probe Southern blots. Unfortunately, such single-locus probes or primers currently have to be developed a new for each species. In a preliminary stage of the work, the efficiency of primers in terms of production of readable PCR products and polymorphism will be tested and the PCR conditions will be optimised. At least 30 primers will be designed for the regions flanking the microsatellite repeat cores. The efficiency of primers in terms of production of readable PCR products and polymorphism will be tested on a pooled set of 100 individuals from throughout the geographical range, and the PCR conditions will be optimized. At least 5 pairs of primers will be used for routine scoring of individuals. Priority will be given to trinucleotide repeats. All 20 samples (100 specimens each) collected during the first year of the project will be scored for the developed microsatellite loci.
Moreover, by combining microsatellite information with that derived from the other types of markers, it is possible that a more complete image of the population dynamics of the species will emerge.

2.4.- Single-Strand ConformationPolymorphism (SSCP) analysis
Single-Strand Conformation Polymorphism (SSCP) is a molecular technique characterized by a high sensitivity. It has indeed the power to detect single point mutations, which gives it more resolution than RFLPs, in spite of its plainness of use. These features make SSCP a powerful tool for the detection and analysis of DNA variation and one of the most used approaches for population genetic analysis. Moreover polymorphisms detected by SSCP segregate as co-dominant markers, unlikeRAPDs, and can be therefore used to test for random mating in populations. SSCP will be applied on a significant number of horse mackerel individuals from all the 20 sampling localities of the project. Aim of this approach will be to analyse at a fine scale the genetic structure of the species and to provide data about the degree of mating isolation between the detected stock-units.

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