Molecular marker techniques

All living things are made up of cells that are programmed by genetic material called DNA. The DNA molecule is made up of a long chain of nitrogen-containing bases (there are 4 different bases - A, C, G and T). Only a small fraction of the DNA sequence typically makes up genes, which code for proteins, while the remaining and major share of the DNA represents non-coding sequences whose role is not yet clearly understood. The genetic material is organised into sets of chromosomes (e.g. 5 pairs in Arabidopsis thaliana; 30 pairs in cattle), and the entire set is called the genome. In a diploid individual (i.e. where chromosomes are organised in pairs), there are two alleles of every gene - one from each parent.

Prior to sexual reproduction, the DNA on the chromosomes in the sex cells (pollen/ sperm and ovule/egg) will undergo some mixing, or recombination so that different segments of the chromosome will come from different parents. This will create new combinations of genes and characteristics in the offspring. Importantly, genes and sections of DNA which are physically close together on the chromosome will be more likely to remain linked together. This occurs naturally in plants and animals, including humans.

During conventional breeding, breeders will cross two parent lines and select the offspring which have the most favourable combinations of characteristics. Breeders can map the relative positions of genes controlling visible characteristics on the chromosomes. Such a genetic map can help them to identify markers linked to traits that are selected for.

However, some traits are very difficult to select for. For example, complex traits such as seed size may be controlled by a large number of genes that are not always inherited together. Other characteristics, such as high vitamin content, may not be visible and may require complex biochemical analysis of the plant material. Further traits may only be detectable in response to damaging treatments or at particular times in the life cycle. Therefore, selecting for drought tolerance or disease resistance requires the breeder to inflict drought or disease on the crops at each generation. This may be complicated, time consuming and difficult to control such that if the drought or infection is too extreme the crops will die. Similarly, to screen for seed related characteristics, it would be necessary to grow all the offspring throughout their life-cycle until they fruit: this incurs substantial costs in terms of time and space, especially for trees.

Molecular Markers

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Molecular markers are identifiable DNA sequences, found at specific locations on the chromosomes, and transmitted by the standard laws of inheritance from one generation to the next. They may be located in or near genes. Since DNA is the same in every cell, the molecular markers can be identified by a DNA test regardless of the developmental stage, age, or environmental challenges experienced by the organism. Molecular Markers can be useful tools to both facilitate breeding programmes (in Marker Assisted Selection - MAS) or to aid in characterisation of collections of germplasm (or varieties). In breeding, crop scientists can map which DNA markers are linked to particular phenotypic characteristics and use this information to accelerate and facilitate breeding programmes. For example, if it is known that two DNA markers are located on either side of a gene that confers drought resistance to adult rice, then breeders can employ a quick DNA test on large numbers of seedlings. If the test shows that the markers are both present, then it is probable that the adult rice plants contains the gene and would display drought tolerance. The breeders could therefore discard most of the seedlings and grow only those individuals containing the markers. Further, it may not be necessary to inflict drought on all plants at every generation, since the presence of the markers should indicate that the plants are tolerant.

Different kinds of molecular markers exist, such as RFLPs, RAPDs, AFLPs, microsatellites and SNPs. They may differ in a variety of ways - such as their technical requirements; the amount of time, money and labour needed; the number of genetic markers that can be detected throughout the genome; and the amount of genetic variation found at each marker in a given population. The information provided by the markers for the breeder will vary depending on the type of marker system used. Each one has its advantages and disadvantages and, in the future, other systems are also likely to be developed. A brief overview of the major marker systems follows:

a) RFLPs: Restriction Fragment Length Polymorphisms are markers detected by treating DNA with restriction enzymes (enzymes that cut DNA at a specific sequence). For example, the EcoR1 restriction enzyme cuts DNA whenever the base sequence GAATTC is found. Differences in the lengths of DNA fragments will then be seen if, for example, the DNA of one individual contains that sequence at a specific part of the genome (e.g. tip of chromosome 3) whereas another individual has the sequence GAATTT (which is not cut by EcoR1). RFLPs were the first molecular markers to be widely used. Their use is, however, time-consuming and expensive and simpler marker systems have subsequently been developed.

b) RAPDs: Random Amplified Polymorphic DNA markers were first described in 1990. They are detected using the polymerase chain reaction (PCR), a widespread molecular biology procedure allowing the production of multiple copies (amplification) of specific DNA sequences. The analysis for RAPD markers is quick and simple, although results are sensitive to laboratory conditions.

c) AFLPs: In the mid 1990's, another PCR-based method of generating molecular markers was described, giving rise to Amplified Fragment Length Polymorphism (AFLP) markers. With this technique, DNA treated with restriction enzymes is amplified with PCR. It allows selective amplification of restriction fragments giving rise to large numbers of useful markers which can be located on the genome relatively quickly and reliably. Unlike other methods described here, the technique is patented.

d) Microsatellites: also known as Simple Sequence Repeats (SSRs) are simple DNA sequences (e.g. AC), usually 2 or 3 bases long, repeated a variable number of times in tandem. They are easy to detect with PCR and a typical microsatellite marker has more variants than those from other marker systems. Initial identification of microsatellites is time-consuming.

e) SNPs: in recent years, Single Nucleotide Polymorphisms (SNPs), i.e. single base changes in DNA sequence, have become an increasingly important class of molecular marker. The potential number of SNP markers is very high, meaning that it should be possible to find them in all parts of the genome, and micro-array procedures have been developed for automatically scoring hundreds of SNP loci simultaneously at a low cost per sample.

Korzun (2003) considering the case of cereals, provided a comparison of these marker systems:

Feature	RFLPs	RAPDs	AFLPs	Microsats	SNPs
Amount of DNA required (μg)	10	0.02	0.5-1.0	0.05	0.05
Quality of DNA required	high	high	moderate	moderate	high
PCR-based	no	yes	yes	yes	yes
Number of polymorphic loci analysed per analysis	1.0-3.0	1.5-50	20-100	1.0-3.0	1.0
Ease of use	not easy	easy	easy	easy	easy
Amenable to automation	low	moderate	moderate	high	high
Reproducibility	high	unreliable	high	high	high
Development cost	low	low	moderate	high	high
Cost per analysis	high	low	moderate	low	low

Some informative diagrams describing the different marker techniques can be found at the link below.

http://www.igd.cornell.edu/MolecularMarkers/Sequence-tagged%20sites.pdf

Marker Assisted Selection

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Marker Assisted Selection (MAS) is based on the identification and use of markers which are linked to the gene(s) controlling the trait of interest. By virtue of that linkage, selection may be applied to the marker itself. The advantage consists in the opportunity of speeding up the application of the selection procedure. For instance, a character which is expressed only at the mature-plant stage, may be selected at the plantlet stage if selection is applied to a molecular marker. Also, selection may be applied simultaneously to more than one character, and selection for a resistance gene can be carried out without needing to expose the plant to the pest, pathogen or deleterious agent. Finally, when there is linkage between a molecular marker and a quantitative trait locus (QTL), selection may become more efficient and rapid. The construction of detailed molecular and genetic maps of the genome of the species of interest is a prerequisite for most forms of MAS. However, the current cost of the application of these techniques is significant, and the choice of one technique rather than others may be dictated by cost factors. There are still very few examples of crop varieties in farmers' fields in developing countries which have been developed based on MAS, largely because of the currently prohibitive cost of incorporating large-scale MAS into the budgets of most plant breeding programmes.

Molecular markers for germplasm characterisation

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In order to improve crops or adapt them to respond to new challenges posed by changes in the environment, diseases or the market, there need to be plant genetic resources to include in breeding experiments. Crop genetic resources include farmer's varieties and landraces, elite and special material and crop varieties developed by plant breeders and other researchers, wild and weedy relatives of crop plants, and wild plants harvested for food. Scientists and breeders are collecting and storing these genetic resources or germplasm as insurance for the future and for use in plant breeding programmes today.

Due to the costs involved in storage and maintenance, it is important to know about the genetic variation contained within a germplasm collection. This can help ensure that more unusual (and therefore potentially valuable) varieties are kept while duplicates may be discarded to save time, space and money. Further, when sampling the germplasm collections for use, it is more efficient to select those lines that are representative of their populations.

It is not prudent to rely only on visible morphological observations or protein markers to determine variation in a germplasm collection. This is because visible and protein markers may be affected by age, developmental stage or the environmental conditions in which the crop samples are grown.

Examples from FAO-BioDeC: Molecular marker-related research activities in Africa are reported to be underway in only a few of the countries, such as Ethiopia, Nigeria, South Africa and Zimbabwe; the range of African crops under study with molecular markers, however, is very wide: from traditional commodities to tropical fruits.

Other Documents

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Some interesting documents on the use of Molecular Markers can be found below.

Dreher, K., Morris, M., Khairallah, M., Ribaut, J.M., Pandey, S. & Srinivasan, G. 2000. Is marker-assisted selection cost-effective compared to conventional plant breeding methods? The case of quality protein maize. Paper presented at the 4th ICABR Conference on Economics of Agricultural Biotechnology, Ravello, Italy, 24-28 August 2000.

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