Examples of Molecular Markers | Plant Genetics

Molecular markers are DNA sequences, whose inheritance pattern can be established. Classic examples of molecular markers are: 1. Restriction Fragment Length Polymorphism (RFLP) 2. Randomly Amplified Polymorphic DNA (RAPD) 3. Amplified Fragments Length Polymorphism (AFLP) 4. Sequence Characterized Amplified Region (SCAR).

Example # 1. Restriction Fragment Length Polymorphism (RFLP):

This novel non-PCR based approach is based on hybridization of probe to fragments of genomic DNA following digestion with restriction enzymes. The polymorphic nature of DNA was first elucidated by RFLP technology. Genetic diversity among plant species is mainly due to the variation in the DNA sequence, while ascertaining the polymorphic nature of the genetic material in which genetic information is stored in the DNA sequence.

The genomic DNA from the sample being tested is digested with restriction endonucleases. This enzyme cut DNA at specific sites with sequences known as restriction enzyme recognition sequences. The restric­tion digestion resultant DNA fragments are separated by electrophoresis on an agarose gel and subjected for southern blotting technique.

It blots DNA from gel to a nylon membrane to allow hybridization with probe. The pre-labelled probe is used to hybridize with blotted DNA. Suit­able DNA probe is usually from the species being studied. Labelled probes are mainly a genomic clones are cDNA. Sometimes ribosomal RNA genes (multiple copy genes) are useful for analysis.

Radiolabeled and non-radiolabelled probes are available in this process. The labelling of probes was traditionally carried out using radioisotopes. The non-radioactive labelled ones are biotin digoxigen as fluorescence material. The nature of hybridization pattern reveals polymorphism in the DNA sample and exhibit sequence difference between individuals.

Although replication of DNA proceeds accurately several agents within the cell is responsible for alteration in se­quence. Single base pair changes takes place in the DNA could be responsible for sequence alteration or certain chromosomal abberations like translocation, invertion, and deletion could be mainly attributed to large amount of variation.

Either low or high profiles changes in the DNA sequence consequently result in the loss or gain of recognition site. The restriction enzymes, suppose to cut sequence fails to do so due to alteration with in recognition sequence which in turn lead to restriction fragments of different length. Thus, one DNA fragments obtained using one specific restriction enzyme is considered as one RFLP.

Distribution of altered nucleotide through­out the genome sequence results in restriction fragments of different length between genotypes and could be detected on southern blot followed by hybridization with suitable probes. For exam­ple, restriction enzyme cut the DNA sequence at specific sequence.

Loss of one or more nucleotide in the restriction recognition sequence result in the lack of recombination and EcoRI enzyme fail to cleavage DNA strands at the altered site. As a result EcoRI generated larger DNA fragments. In this fashion presence of RFLP in the chromosomes of two homologous chromosomes can be detected based on the presence of restriction site in the DNA molecule of one chromosome which produces shorter fragments and lack of cut (restriction) site in other homologous chromosome produces longer fragments. Thus, it is possible to distin­guish two chromosomes in such individuals based on this RFLP.

In addition to chromosomal alteration repeated sequence of 9 to 65 base pairs long that occur a variable number of times (10-300 times) may also provide valuable level of polymorphism in higher organism, which can be easily detected by hybridization. This special case of RFLP analysis has been referred to as VNTR (variable number of tandem repeats) analysis.

These VNTR sequences are being considered as valuable markers, otherwise known as minisatellites and acts as valuable tool in distinguish the genotype of plants such as rice (Oryza sativa) and bean (Phaseolus vulgaris). These minisatellites markers can be studied using probes generated by PCR.

RFLP requires large quantities of high quality DNA for detection of single copy loci and detects only a fraction of existing sequence variability. Discovery of polymerase chain reaction (PCR) provides additional approach to analysis, which can overcome required quantity of DNA. The PCR can amplify specific region of DNA obtained. The amplified sequence can be compared by RFLP directly on stained agarose gel without entering into the southern hybridization.

Fig. 24.1A. Overview of RFLP technique.

Example # 2. Randomly Amplified Polymorphic DNA (RAPD):

These are dominant markers, discovered in 1990. It is one of the widely used techniques to characterise nature of DNA from plant and other organism. One of the main bases for this technique is to use PCR with short oligonucleotide primers of random sequence. Based on this approach Randomly Amplified Polymorphic DNA (RAPD) markers can be generated.

This technique is also the basis for arbitrarly primed polymerase reaction (AP-PCR) and DNA amplification finger printing (DAF). The principle behind RAPD is the amplification with short primers (9-10 mer) is such that many sites in genomic samples are potential template for primers. Variation in the concentration of primers or template and conditions used in PCR result in the amplification of different products to generate RAPD profiles.

Selection of standard primers, nucleotide, DNA polymorphism type and magnesium concentrations contribute in reproducibility. When genomic DNA from two plants species are subjected for RAPD, often produces different amplification patterns. A particular fragment generated for species but not for other species represents DNA polymorphism based on RAPD profiles. This difference is the basis and can be used as genetic marker or RAPD marker.

Advantages of RAPD over RFLP are that it requires crude extract of DNA. However, in RFLP, requires relatively pure DNA. Even small amount of DNA at nanogram level (5-20 ng) is sufficient for RAPD. In addition, use of radio isotopes is not essential and whole genome can be surveyed using random primers.

Its automation is completely and comparatively easy and exhibit intermediate in reliability. RAPD are efficient in analysis of less known species because they can be applied without prior knowledge of gene sequence. RAPD can be performed similar to those of polymerase chain reaction using genomic DNA from the species of interest and random primers.

Random primers are commercially available and they can be prepared by different combinations of nucleotides. These primers are synthesised based on choosing random sequence of the DNA. Each random primer when used will anneal to a various regions of the DNA and possible different loci can be analysed.

The RAPD method requires use of agarose gel to analyse the PCR products, for example, DNA sequence between two plant variety and individual can be distinguished by their primer binding site. Binding of one particular primer to one variety fail to bind to other due to lack of binding site, which results in the lack of particular band among PCR amplified product.

RAPD method can be refined to reveal more polymorphism if combined with restriction digestion, for example, certain economically important cereal crop like wheat exhibit very little genetic variation. Extracted DNA from wheat sample is first digested with restriction enzyme before subjected for RAPD, which reveals more refined DNA polymorphism.

Example # 3. Amplified Fragments Length Polymorphism (AFLP):

Amplified fragments length polymorphism (AFLP) is a PCR-based technique that involved restriction digestion of genomic DNA followed by ligation of adaptors to DNA fragments generated and consequently follows selective PCR amplification of these fragments.

DNA from plant to be analysed is digested with a restriction nucleases and short double stranded adapters (short oligonucleotides) are ligated to the ends of DNA fragments. The purpose of adding adaptors is because sequence of adaptors and adjacent restriction sites serve as primer binding sites for further amplification.

These restriction endonuclease sites are then amplified by PCR using primers complementary to the added adaptors and restriction sites. The degree of additional specificity can be (Fig. 24.1B) provided by a few specific nucleotide attached to 3′ and of the PCR primer. The choice of enzymes and primer length is crucial for maximum results in difficult application.

Fig. 24.1 Overview of AFLP technique.

The amplified fragments are separated on an agarose gel, subjected for southern blot and can be visualised by autoradiography or fluorescent sequencing equipment. The level of polymorphism detected by AFLP is lower than other techniques like microsatellites.

The AFLP technique is however, enable in analysing a large number of polymorphic loci simultaneously using single primer combination on one gel provides valuable information over other mapping methods. Research kits for labelling AFLP primers are manufactured under licence from key gene and life technology USA.

Example # 4. Sequence Characterized Amplified Region (SCAR) and Sequence Tagged Sites (STS):

Sequence characterized amplified regions (SCAR) is a PCR based method developed by sequencing markers amplified in arbitrary primers experiments can be converted into SCARs. Amplified RAPD products are cloned, sequenced and primer sequence are ascertained from the end of band is identified as RAPD markers.

In SCAR Method, designing of longer primers are required to offer greater degree of specificity. The sequence of the amplified product is used to design longer primers (Fig. 24.3). Thus, longer primers with increased specificity can amplify single repeatable band. Better assessment of F₂ individual can be accomplished by converting dominant RAPD to codominant SCAR.

Reproducible potential of SCAR with longer primer has advantage over short primer used for RAPD analysis. Similarly, RFLP can be converted into SCAR by sequencing two ends on the genomic DNA and primers are designed based on end sequences. The polymorphic regions could be amplified by PCR in which primers were used directly on genomic DNA. Absence of amplified fragment length polymorphism proceeds with restriction digestion of PCR fragments to detect RFLP within the amplified fragments.

STS are short stretches of unique DNA sequence that can be amplified PCR from genomic library or genomic DNA using specific oligonucleotide primers. The STS primers can be designed by sequencing track of genomic RFLP markers, which are further used to develop the primers.