Genetic Mapping

Sampling

Two maternal half-sib families of n=92 each are used as the mapping families. Sampling was conducted in August 2004 at 61 days post first feeding; which is near the end of metamorphosis and the beginning of weaning from live artemia to pellet feed.

Genetic markers

Sequencing a genome is very expensive and time consuming. A practical alternative is to use genetic markers, such as microsatellites (fig. 1), AFLP's (fig. 2) and SNP's to create a genetic map. Genetic markers are small pieces of DNA (100-400bp) that are highly variable among individuals and are used not only for genetic mapping but forensics, paternity testing and population studies. The genetic map currently consists of approximately 200 microsatellites and 350 AFLP markers. Microsatellites and AFLP's are run on the LiCor 4200 Long Reader and analysed using the Saga GTII software (LiCor, Lincoln, NB. http://www.licor.com/bio/ ) additional microsatellites were analysed using the ABI377 and Genotyper software (Applied Biosystems, Foster City, CA http://www.appliedbiosystems.com/).

Figure 1 Sample microsatellite gel image generated using the LiCor 4200 Long Reader System. Dam is outlined in pink with alleles 1 and 4, sire is outlined in blue with alleles 2 and 3. Progeny are to the left of the size standard (bp). Progeny inherit one allele from each parent (e.g., each progeny exhibits either the 2 or the 3 allele from the sire).

Figure 2 Sample Amplified Fragment Length Polymorphism (AFLP). The dam is outlined in pink and the sire is outlined in blue. AFLP’s are dominant markers, meaning that they are only informative in one parent. The informative parent is heterozygous for a band (indicated with a 2) and the other parent is homozygous for a lack of a band (indicated with a 1). Progeny with a 2 allele have inherited the restriction site from the heterozygous parent and progeny with a 1 allele have inherited the lack of a restriction site from the homozygous parent.

Linkage Mapping

Using software to examine the segregation patterns among hundreds of genetic markers in the progeny from a given family we can determine the crossover or recombination frequency between two markers (where 1% recombination = 1cM (centiMorgan)). When the recombination frequency is very low between two markers, we know those markers are very close together on a chromosome. When we combine the analysis of hundreds of markers we are able to place them in the correct order and the correct distance apart along each chromosome (fig 3). Mapping analysis is conducted using the LINKMFEX software package developed by Roy Danzmann (University of Guelph, http://www.uoguelph.ca/~rdanzman/software/LINKMFEX/).

Quantitative Trait Analysis

Using a genetic map, and powerful statistics we can detect genes very close to genetic markers that affect traits of importance such as growth or disease resistance (known as Quantitative Trait Loci (QTL)). Several traits have been measured in the experimental families including body weight, length, width, myotome height, pigmentation and eye migration (fig.4). Several software packages are used in this analysis including QTL Cartographer (Z.B.Zeng et al. Statistical Genetics, North Carolina State Univeristy, http://statgen.ncsu.edu/qtlcart/) and QTL Express (Seaton et al. University of Edinburgh, UK, http://qtl.cap.ed.ac.uk/).

Figure 3: Sample linkage groups from halibut (Hippoglossus hippoglossus). Distance along the linkage group is shown in cM. Markers include microsatellites from Atlantic halibut (Hhi) and Japanese flounder (Poli) and AFLP (e.g., AAC/CAC311). QTL are identified as a line (blue = trait 1; red = trait 2) spanning the 95% CI in cM with the percent variance explained, indicated by the arrow. For example the QTL identified on Linkage Group 1 accounts for 17.9% of the trait 1 variance in the experimental families and the 95% CI spans approximately 16cM.

Figures 4a-c Morphometric data collected for a quantitative trait analysis in Atlantic halibut (H. hippoglossus). All pictures are of the same fish.

(4a) Head-on view of eye migration measurement in degrees. A properly migrated eye was around 70° whereas a non-migrated eye would remain near 180°, a diagramatic inset is provided for clarity

(4b) Side-view used to measure fork-length, maximum width and myotome height

(4c) approximation of the method used to evaluate % surface area pigmented (%SA), in this example %SA » 60%. The true analysis using Simple PCI v4.0 Imaging Software (Compix Inc) is a comparative method based on a preset level of complete and non-existent pigmentation and the difference from each of these extremes is measured as a whole