Markers

Types of genetic markers

Genetic markers can be divided into three types:

• Morphological markers
• Biochemical markers
• DNA (or molecular) markers

Morphological markers

Morphological markers are the first markers that were used in plant breeding. They are visible in the phenotype of an individual organism. When the trait of interest is located closely to the locus of the morphological marker, this marker can be used for MAS. In any parental pair, used in a crossing, we can only use the morphological traits for which these two parents differ. This may be hairiness of leaf base, color of flower, some leaf shape aspect, leaf tip necrosis etc. However, such morphological markers do not usually cover the entire genome, which limits their use. Besides, in modern cultivars, the morphological aspects that differ between pairs of cultivars is very limited, and hence the number of possible morphological markers is also very limited. Further, several of such markers can only be judged in advanced stages of plant development, e.g. flower colour. So, an early and rapid screening (i.e. in the seedling stage) is not possible, since we should wait until the population to be judged is flowering in order to use flower color as a marker.

Biochemical markers

Biochemical markers are allelic variations of enzymes, called isozymes. Different isozymes of a specific enzyme have the same function in an organism. Isozymes differ slightly in structure (size, electrical charge, three-dimensional structure) which cause them to move at a different speed through an electrophoresis gel. These detectable differences are the basis for biochemical markers. Just as morphological markers, different isozymes can be used to select for desired traits if they are linked to genes of interest. 

For biochemical markers, the choice is also limited. There is no infinite number of enzyme systems, and for each enzyme system, the parental pair should be checked whether they have in any of their organs and development stages different isozymes. Each isozyme scoring requires a separate analysis. So, although the number of potential biochemical markers is much larger than morphological markers, their application is cumbersome and it is hard to find enough biochemical markers to cover the whole genome of the plant. Since the development of DNA-based markers, they are hardly used anymore.

DNA (or molecular) markers

DNA-based ("molecular") markers to be applied for selection in a population derived from a cross between two parents are based on variation in sequences in DNA. For example, for a cross of two homozygous parents, it is based on variation between alleles of one parent versus those of the other parent. For a cross where one or both parents are heterozygous, it is based on variation between the two alleles within the same parent(s).

Even small differences in DNA sequence may be visualized to become a marker. Since the DNA consists of a huge amount of DNA base pairs, within most crops a great amount of variation accumulated over time. Most of the first generation DNA markers appeared in non-coding regions of DNA, which are selectively neutral areas of DNA. Nowadays, the aim has shifted to markers that are based on DNA sequence differences inside genes. Since all DNA variation inside and outside genes can be used to develop a marker, the number of DNA markers that can be developed is in principle almost infinite, as long as there is variability across different genotypes at many places in the genome.

An additional advantage of DNA markers is that DNA occurs in each plant cell, and all plant cells (in principle) of an individual carry the same DNA. So, any plant cell or tissue is as good as the other to extract DNA, and we do not have to wait until plants developed to maturity before we can isolate DNA to visualize the markers. Use of DNA markers does have limitations as well. These will be discussed later on. 

Development of DNA markers

DNA markers are visualizations of sequence differences in the DNA. These visualizations can be realized in different ways. The older marker types were developed using various tools and principles. Often, the different procedures involve techniques such as:

1. Cutting the DNA at the restriction sites using restriction enzymes.
2. Amplifying the DNA fragments by PCR, resulting in numerous copies of this fragment: the amplification product. This could be an amplification with rather short primers to obtain many fragments or long primers based on knowledge of a DNA sequence of interest (mostly of a gene).
3. Separation of the amplified products by fragment size through gel electrophoresis and staining with chemicals (ethidium bromide or silver). This procedure reveals bands of DNA in the gel.

With DNA sequencing techniques becoming increasingly efficient and inexpensive, it is relatively easy to find large numbers of SNPs: single nucleotide polymorphisms. This marker type is based on differences of only one base pair between parents.

DNA of a genotype that is heterozygous for a C/T SNP polymorphism, highlighted in red. The SNP is called after the forward DNA strand base (5' strand).

High throughput sequencing methods now allow the generation of many DNA sequence fragments. Several software programs have been developed to compare the generated sequences ('reads') across a number of accessions and to find SNPs that are polymorphic between accessions. Even for species of which no reference genome is available, this sequencing and the selection of SNPs can be done.

Types of markers compared to each other

Effect of developmental stage

Many traits, such as fruit size, become visible only at a certain developmental stage. However, the genes for fruit size (and DNA markers associated with these genes) are already present from the moment of fertilization. As soon as the plant is big enough to harvest a part of the plant (a small leaf will be enough), one can screen the plant material for selected parts of the DNA. The independence of developmental stage is an important advantage of molecular markers above morphological markers.

Summary

→    Three different types of genetic markers exist: those that are detected morphologically, biochemically or by DNA analysis

→    Each type of genetic marker is analyzed visually; only the visualization method used differs 

→    DNA markers exist in almost infinite numbers, they are not affected by the environment, and they can be determined in any developmental stage