DNA MARKERS FOR GENETIC MAPPING

BLS 308

HOZA, A.S

DNA marker:refers to a specific DNA variation between

individuals that has been found to be associated with a certain characteristic (e.g. increased tenderness).

These different DNA or genetic variants = known as alleles.

DNA marker testing or genotyping determines which alleles an animal is carrying for a DNA marker(s).

Use of this genotype information from DNA marker tests associated with simple traits - straight forward.

Such traits - controlled by a single gene and a marker allele associated with that gene can perfectly predict the phenotype of that trait (the physical attributes of an animal).

DNA tests for simple traits include those for certain diseases:

DUMPS (Deficiency of Uridine Monophosphate Synthase)

BLAD (Bovine Leukocyte Adhesion Deficiency)coat color, and horned status.

NB: Most economically relevant traits (ERT) are complex: Controlled by many genes

Influenced by the environment.

Examples of complex traits : growth traits, carcass characteristics and reproductive performance.

Any single DNA marker is associated with only one of the many genes that control complex traits.

1. Restriction fragment length polymorphisms(RFLP)

2. RAPD-Random amplified polymorphic DNA(RAPD)

3. Microsatellite, simple sequence repeat (SSR)markers or short tandem repeat (STR)

4. Single nucleotide polymorphisms (SNPs)

5. Amplified fragment length polymorphism (AFLP)

Types of DNA Markers

A molecular marker based on the differential hybridization of cloned DNA to DNA fragments in a sample of restriction enzyme digested DNAs.

The marker is specific to a single clone/restriction enzyme combination

Restriction Fragment Length Polymorphism (RFLP)

RFLP markers have several advantages

1) They are codominant and unaffected by the environment.

2) Any source DNA can be used for the analysis.

3) Many markers can be mapped in a population that is not stressed by the effects of phenotypic mutations.

Variations occurring within a species in the length of DNA fragments generated by a specific endonuclease.

A molecular marker based on the differential PCR amplification of a sample of DNAs from short oligonucleotide sequences

2. Randomly Amplified Polymorphic DNA (RAPD)

The primary drawback to RAPD markers is that they are dominant and do not permit the scoring of heterozygous individuals.

3. Microsatellite Markers

1. Short segments of DNA that have a repeated sequence such as CACACACA, and they tend to occur in non-coding DNA.

In some microsatellites, the repeated unit (e.g. CA) may occur four times, in others it may be seven, or two, or thirty.

2. The most common way to detect microsatellites is to design PCR primers that are unique to one locus in the genome and that base pair on either side of the repeated portion.

Therefore, a single pair of PCR primers will work for every individual in the species and produce different sized products for each of the different length microsatellites.

5. AFLP- Amplified fragment length polymorphism

DNA fingerprinting technique that combines features of RFLP and RAPD techniques for amplification of a subset of genomic restriction fragments using selective primers

A molecular marker generated by a combination of restriciton digestion and PCR amplification

A molecular marker system based on the staining of proteins with identical function, but different electrophoretic mobilities

6. Isozyme

Isozyme LociIsozymes are protein markers.

The technique is based on the principal that allelic variation exists from many different proteins.

Example, alleles of malic dehydrogenase would both perform the correct enzymatic function, but the electrophoretic mobility of the two may differ.

Therefore, two alleles would not migrate to the same location in a starch gel.

The procedures to identify isozyme variation : A crude protein extract is made from some tissue sources, usually leaves.

The extracts are next separated by electrophoresis in a starch gel.

The gel is then placed in a solution that contains reagents required for the enzymatic activity of the enzyme you are monitoring.

In addition, the solution contains a dye that the enzyme can catalyze into a color reagent that stains the protein.

In this manner allelic variants of the protein can be visualized in a gel.

First, the number of isozyme loci that can be scored is limited.

To date, only 40-50 reagent systems have been developed that permit the staining of a particular protein in a starch.

Not all of these reagent systems work efficiently with all plant species. Therefore, for many species only 15-20 loci can be mapped.

A second drawback is tissue variability. Some isozymes are better expressed in certain tissues such as

roots, whereas other are best sampled in leaf tissue.

Therefore, several samplings of the segregating population are necessary to score all the available isozyme.

Because neither of these drawbacks affect RFLP or RAPD loci, isozyme loci are rarely scored today.

Several drawbacks of isozyme.