Molecular Markers

A genetic marker is any character that can be measured in an organism which provides information on the genotype (i.e. genetic make-up) of that organism. A genetic marker may be a recognizable phenotypic trait (e.g. height, colour, response to pathogens), a biochemical trait (e.g. an isozyme) or a molecular trait (i.e. DNA based).Whereas phenotypic markers depend on expression of genes and are limited to those genes expressed at a particular time or under particular developmental or environmental conditions, DNA-based markers provide an almost unlimited supply of markers that identify specific sequences across the genome. Their advantages are:
(i) Single base changes in DNA can be identified, providing many potential marker sites across a genome.
(ii) They are independent of developmental stage, environment or expression.
(iii) Markers can be found in non-coding or repetitive sequences.
(iv) Most DNA marker sequences are selectively neutral.
Thus, for example, because about 80% of the wheat genome is noncoding DNA, only molecular markers can be used to identify polymorphisms and to map ‘loci’ in these regions of the genome.
Types of Molecular Markers
There are many potential approaches to identify molecular markers. Most are based on using the polymerase chain reaction (PCR) to amplify specific DNA sequences.3–5 They include:
(i) RFLPs (restriction fragment length polymorphisms);
(ii) RAPD-PCR (random amplified polymorphic DNA);
(iii) microsatellites or simple sequence repeats (SSRs);
(iv) AFLP (amplified fragment length polymorphisms).
RFLPs rely on the combination of a probe and restriction enzymes to identify polymorphic DNA sequences using Southern blotting. This approach requires either radioactive or non-radioactive detection methods to identify polymorphic DNA bands and is therefore more time
consuming than PCR-based methods.

RAPD-PCR does not require sequence information and involves amplifying random pieces of DNA in which PCR is primed by a single 10 base primer at low stringency, such that random sequences of DNA are amplified based on homologous sequences to the primer being present in the target DNA. It is a useful initial approach to identify polymorphisms, but is not regarded as reproducible enough between laboratories.
Microsatellites or SSRs are groups of repetitive DNA sequences that are present in a significant proportion of plant genomes. They consist of tandemly repeated mono-, di-, tri-, tetra- or pentanucleotide units. The number of repeats varies in different individuals and so the different
repeats can be regarded as ‘polymorphic’ alleles at that ‘locus’. To reveal polymorphic microsatellite sequences, it is necessary to sequence the conserved flanking DNA and to design PCR primers that will amplify the repeat sequences. (Because of the repetitive nature of the amplified sequences, typically the main amplified PCR band and additional ‘stutter’ bands are generated.) For example, at microsatellite locus Hspl76 of soybean, there is an AT repeat with 13 different numbers of bases in the repeated units in different soybean accessions.Microsatellites provide reliable, reproducible molecular markers.
AFLP is also a PCR-based technique, in which selective pre-amplification and amplification steps are carried out to amplify a subset of fragments of the genome, depending on the linkers added and primers used. Many potentially polymorphic fragments are generated by this approach. Polymorphic bands between parents can be identified and linked to useful traits.
Both microsatellite and AFLP markers can be analysed using autoradiography or a DNA sequencer, using fluorescent tags. The latter allows multiplexing such that three different coloured tags plus a size marker can be run in each lane. A single multiplexed AFLP gel can generate 100 polymorphic bands.