Marker-assisted selection

Marker assisted selection or marker aided selection is an indirect selection process where a trait of interest is selected based on a marker linked to a trait of interest, rather than on the trait itself. This process has been extensively researched and proposed for plant- and animal- breeding.
For example, using MAS to select individuals with disease resistance involves identifying a marker allele that is linked with disease resistance rather than the level of disease resistance. The assumption is that the marker associates at high frequency with the gene or quantitative trait locus of interest, due to genetic linkage. MAS can be useful to select for traits that are difficult or expensive to measure, exhibit low heritability and/or are expressed late in development. At certain points in the breeding process the specimens are examined to ensure that they express the desired trait.

Marker types

The majority of MAS work in the present era uses DNA-based markers. However, the first markers that allowed indirect selection of a trait of interest were morphological markers. In 1923, Karl Sax first reported association of a simply inherited genetic marker with a quantitative trait in plants when he observed segregation of seed size associated with segregation for a seed coat color marker in beans. In 1935, J. Rasmusson demonstrated linkage of flowering time in peas with a simply inherited gene for flower color.
Markers may be:

Morphological These were the first markers loci available that have an obvious impact on the morphology of plants. These markers are often detectable by eye, by simple visual inspection. Examples of this type of marker include the presence or absence of an awn, leaf sheath coloration, height, grain color, aroma of rice etc. In well-characterized crops like maize, tomato, pea, barley or wheat, tens or hundreds of genes that determine morphological traits have been mapped to specific chromosome locations.
Biochemical A protein that can be extracted and observed; for example, isozymes and storage proteins.
Cytological Cytological markers are chromosomal features that can be identified through microscopy. These generally take the form of chromosome bands, regions of chromatin that become impregnated with specific dyes used in cytology. The presence or absence of a chromosome band can be correlated with a particular trait, indicating that the locus responsible for the trait is located within or near to the banded region. Morphological and cytological markers formed the backbone of early genetic studies in crops such as wheat and maize.
DNA-based Including , restriction fragment length polymorphism, random amplification of polymorphic DNA, amplified fragment length polymorphism, and single nucleotide polymorphisms.
Positive and negative selectable markers

The following terms are generally less relevant to discussions of MAS in plant and animal breeding, but are highly relevant in molecular biology research:

Positive selectable markers are selectable markers that confer selective advantage to the host organism. An example would be antibiotic resistance, which allows the host organism to survive antibiotic selection.
Negative selectable markers are selectable markers that eliminate or inhibit growth of the host organism upon selection. An example would be thymidine kinase, which makes the host sensitive to ganciclovir selection.

A distinction can be made between selectable markers and screenable markers. Most MAS uses screenable markers rather than selectable markers.

Gene vs marker

The gene of interest directly causes production of protein or RNA that produce a desired trait or phenotype, whereas markers are genetically linked to the gene of interest. The gene of interest and the marker tend to move together during segregation of gametes due to their proximity on the same chromosome and concomitant reduction in recombination between the marker and gene of interest. For some traits, the gene of interest has been discovered and the presence of desirable alleles can be directly assayed with a high level of confidence. However, if the gene of interest is not known, markers linked to the gene of interest can still be used to select for individuals with desirable alleles of the gene of interest. When markers are used there may be some inaccurate results due to inaccurate tests for the marker. There also can be false positive results when markers are used, due to recombination between the marker of interest and gene. A perfect marker would elicit no false positive results. The term 'perfect marker' is sometimes used when tests are performed to detect a SNP or other DNA polymorphism in the gene of interest, if that SNP or other polymorphism is the direct cause of the trait of interest. The term 'marker' is still appropriate to use when directly assaying the gene of interest, because the test of genotype is an indirect test of the trait or phenotype of interest.

Important properties of ideal markers for MAS

An ideal marker:

Has easy recognition of phenotypes - ideally all possible phenotypes from all possible alleles
Demonstrates measurable differences in expression between trait types or gene of interest alleles, early in the development of the organism
Testing for the marker does not have variable success depending on the allele at the marker locus or the allele at the target locus.
Low or null interaction among the markers allowing the use of many at the same time in a segregating population
Abundant in number
Polymorphic
Drawbacks of morphological markers

Morphological markers are associated with several general deficits that reduce their usefulness including:

the delay of marker expression until late into the development of the organism
allowing dominance to mask the underlying genetics
pleiotropy, which does not allow easy and parsimonious inferences to be drawn from one gene to one trait
confounding effects of genes unrelated to the gene or trait of interest but which also affect the morphological marker
frequent confounding effects of environmental factors which affect the morphological characteristics of the organism

To avoid problems specific to morphological markers, DNA-based markers have been developed. They are highly polymorphic, exhibit simple inheritance, are abundant throughout the genome, are easy and fast to detect, exhibit minimum pleiotropic effects, and detection is not dependent on the developmental stage of the organism. Numerous markers have been mapped to different chromosomes in several crops including rice, wheat, maize, soybean and several others, and in livestock such as cattle, pigs and chickens. Those markers have been used in diversity analysis, parentage detection, DNA fingerprinting, and prediction of hybrid performance. Molecular markers are useful in indirect selection processes, enabling manual selection of individuals for further propagation.

Selection for major genes linked to markers

'Major genes' that are responsible for economically important characteristics are frequent in the plant kingdom. Such characteristics include disease resistance, male sterility, self-incompatibility, and others related to shape, color, and architecture of whole plants and are often of mono- or oligogenic in nature. The marker loci that are tightly linked to major genes can be used for selection and are sometimes more efficient than direct selection for the target gene. Such advantages in efficiency may be due for example, to higher expression of the marker mRNA in such cases that the marker is itself a gene. Alternatively, in such cases that the target gene of interest differs between two alleles by a difficult-to-detect single nucleotide polymorphism, an external marker may present as the most realistic option.

Situations that are favorable for molecular marker selection

There are several indications for the use of molecular markers in the selection of a genetic trait.
Situations such as:

The selected character is expressed late in plant development, like fruit and flower features or adult characters with a juvenile period
The expression of the target gene is recessive
There are special conditions for expression of the target gene, as in the case of breeding for disease and pest resistance. Sometimes inoculation methods are unreliable and sometimes field inoculation with the pathogen is not even allowed for safety reasons. Moreover, sometimes expression is dependent on environmental conditions.
The phenotype is affected by two or more unlinked genes. For example, selection for multiple genes which provide resistance against diseases or insect pests for gene pyramiding.

The cost of genotyping is decreasing thus increasing the attractiveness of MAS as the development of the technology continues.

Steps for MAS

Generally the first step is to map the gene or quantitative trait locus of interest first by using different techniques and then using this information for marker assisted selection. Generally, the markers to be used should be close to gene of interest in order to ensure that only minor fraction of the selected individuals will be recombinants. Generally, not only a single marker but rather two markers are used in order to reduce the chances of an error due to homologous recombination. For example, if two flanking markers are used at same time with an interval between them of approximately 20cM, there is higher probability for recovery of the target gene.

QTL mapping techniques

In plants QTL mapping is generally achieved using bi-parental cross populations; a cross between two parents which have a contrasting phenotype for the trait of interest are developed. Commonly used populations are near isogenic lines, recombinant inbred lines, doubled haploids, back cross and F₂. Linkage between the phenotype and markers which have already been mapped is tested in these populations in order to determine the position of the QTL. Such techniques are based on linkage and are therefore referred to as "linkage mapping".A