Detecting DNA
Polymorphisms

Mapping and
Mapping Populations

Genetics of Mapping
Molecular Loci

Specialized Mapping
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The Classes of Molecular Markers

Linkage maps of many plant species were limited in size until the advent of molecular mapping. The primary difficulty with developing linkage maps was the inability to incorporate many markers into a single stock to be used for genetic analysis. This inability occurred because of the deleterious effects of the expression of all mutant phenotypes in the single stock. Because normal DNA or protein molecules are used to score the genetic material, molecular markers are phenotypically neutral. This is a significant advantage compared to traditional phenotypic markers.

The three most common types of markers used today are RFLP, RAPD and isozymes. Of the three marker types, RFLPs have been used the most extensively. RFLP markers have several advantages in comparison with the RAPD and isozyme markers: 1) they are codominant and unaffected by the environment; 2) any source DNA can be used for the analysis; and 3) many markers can be mapped in a population that is not stressed by the effects of phenotypic mutations. The primary drawback to RAPD markers is that they are dominant and do not permit the scoring of heterozygous individuals. The weakness of isozyme markers is that each of the proteins that are being scored may not be expressed in the same tissue and at the same time in development. Therefore several samplings of the genetic population need to be made.

RFLP - Restriction Fragment Length Polymorphism; 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

RAPD - Randomly Amplified Polymorphic DNA; a molecular marker based on the differential PCR amplification of a sample of DNAs from short oligonucleotide sequences

AFLP - Amplified Fragment Length Polymorphism; a molecular marker generated by a combination of restriciton digestion and PCR amplification

Isozyme - a molecular marker system based on the staining of proteins with identical function, but different electrophoretic mobilities<

RFLP Loci

RFLP analysis is an application of the Southern hybridization procedure. The general principles will be explained here and we will then discuss several papers to obtain a more in depth understanding of the procedure.

Clones, Enzymes and Informative Hybridizations

RFLP markers are defined by a specific enzyme-probe combination. The first step in the analysis is to derive a set of clones that can be used to identify RFLPs. Genomic clones that represent sequences at random are a poor choice as hybridization probes because plant genomes consist of a large percentage of repeated sequences. Thus, many of the clones will contain repeated sequences, and hybridizations with those clones containing repeated sequences generate many hybridization bands that are difficult to analyze genetically.

The two primary sources of these clones for RFLP mapping of plants are cDNA clones and PstI-derived genomic clones. These two clone sources are generally represent expressed genes whice are in low copy number. cDNA clones are DNA copies of expressed genes. PstI clones are based on the suggestion that expressed genes are not methylated. As we saw earlier, GC and GXC methylation is the most prominent form of methylation in plants. The enzyme PstI enzyme is C-methylation sensitive. Therefore, the enzyme will only cut non-methylated sites. If a gene is expressed, then its sequence will not be methylated and will be susceptible to PstI digestion. And because they probably contain expressed sequences, these fragments would have a greater probability of being low copy number.

Once a series of clones are derived, DNA from potential parental genotypes is digested with a series of enzymes and hybridized with the clones. Some of these hybridizations will generate fragments of only one size and are not polymorphic. Other hybridizations will give a distinctive hybridization pattern for each parent. These polymorphisms occur because the sequence of the probe is homologous to restriction fragments of different sizes. Those genotypes that are highly polymorphic are candidates as parents from which a mapping population can be derived.

RAPD Loci

RAPD markers have recently caught the fancy of many individuals in the field of applied plant breeding. This molecular marker is based on the PCR amplification of random locations in the genome of the plant. With this technique, a single oligonucleotide is used to prime the amplification of genomic DNA. Because these primers are 10 nucleotides long, they have the possibility of annealing at a number of locations in the genome. For amplification products to occur, the binding must be to inverted repeats sequences generally 150-4000 base pairs apart. The number of amplification products is directly related to the number and orientation of the sequences that are complementary to the primer in the genome.

Steps of RAPD PCR Amplification of Plant DNA

1. Isolate total DNA from the individuals of interest.

2. Establish the conditions for the amplification of the DNA from your specific species. Several considerations are: MgCl2, primer and dNTP concentrations and the quality and concentration of the target DNA. The goal of RAPD experiments are to compare populations. This requires well to well and run to run consistency. Thus, you need to be sure that each well of the cycler will produce the same result given the same, target DNA, primer and dNTP concentration. Without this control you may not be sure that the amplification polymorphisms are the result of population variability or cycler variability.

3. The conditions used in our lab for the amplification of bean DNA are:

• 25 ng DNA
• 200 mM dNTP
• 200 uM primer
• 1.5 mM MgCl2
• 1X Promega Taq polymerase buffer
• 1 unit Taq polymerase
• Reaction volume: 10 ul overlaid with 35µ l mineral oil

4. The PCR amplification steps are:

• 94oC, 1 min for denaturation<
• 35oC, 30 sec for annealing
• 72oC, 2 min for extension

This profile is performed 45 times and then the products are completed by a single extension for 7 min at 72oC. (All of these conditions are a modification of published procedures.)

5. The amplification products are then separated on a 2% agarose gel, stained and photographed.

6. Variability is then scored as the presence or absence of a specific amplification product.

For RFLP mapping, you first need to determine the clone/restriction enzyme combinations that are polymorphic between the parents of your mapping population. The analgous experiment with RAPD markers is to identify the primer and reaction conditions that are polymorphic between you parents. These primers and conditions will be used to amplify and score the products of your segregating population.

Isozyme Loci

Isozymes are protein markers. The technique is based on the principal that allelic variation exists from many different proteins. For 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 is simple. 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.

Several drawbacks should be noted with regards to isozyme. 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. Furthermore, 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.

AFLP Loci

Amplified Fragment Length Polymorphism (AFLP) is the most recent class of molecular loci. These loci are generated using a procedure that combines restriction digestion and PCR amplification. The power of this procedure is that you can generate a large number of mappable loci with a single amplification. This will help you saturate a region of the genome rather quickly. The drawback is that procedure is a bit time consuming and requires the running of a DNA sequencing gel.

AFLP Procedure

1. Digest sample DNA with restriction enzymes EcoRI and MseI.

You can connect to the following WWW site for more information about AFLPs. URL=http://carnegiedpb.stanford.edu/methods/aflp.html