Molecular Markers

Post date: Jun 19, 2010 5:25:13 PM

By Ashwani Kumar, Professor of Botany, Department of Botany, University of Rajasthan, Jaipur. 302004, India

The development of molecular techniques for genetic analysis has led to a great augmentation in our knowledge of crop genetics and our understanding of the structure and behavior of various crop genomes. These molecular techniques, in particular the applications of molecular markers, have been used to scrutinize DNA sequence variation(s) in and among the crop species and create new sources of genetic variation by introducing new and favorable traits from landraces and related crop species.

Markers can aid selection for target alleles that are not easily assayed in individual plants, minimize linkage drag around the target gene, and reduce the number of generations required to recover a very high percentage of the recurrent parent genetic background. Improvements in marker detection systems and in the techniques used to identify markers linked to useful traits, has enabled great advances to be made in recent years.

Though restriction fragments length polymorphism (RFLP) markers have been the basis for most of the work in crop plants, valuable markers have been generated from random amplification polymorphic DNA (RAPD) and amplified fragments length polymorphism (AFLP). Simple sequence repeats (SSR) or microsatellite markers have been developed more recently for major crop plants and this marker system is predicted to lead to even more rapid advances in both marker development and implementation in breeding programs.

Identification of the markers linked to useful traits has been based on complete linkage maps and bulked segregant analysis. However, alternative methods, such as the construction of partial maps and combination of pedigree and marker information, have also proved useful in identifying marker/trait associations. A revision of current breeding methods by utilizing molecular markers in breeding programs is, therefore, crucial in the present scenario.

Introduction:

A genetic marker is a gene or DNA sequence with a known location on a chromosome and associated with a particular gene or trait. It can be described as a variation, which may arise due to mutation or alteration in the genomic loci, that can be observed. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long one, like minisatellites.

For many years, gene mapping was limited in most organisms by traditional genetic markers which include genes that encode easily observable characteristics such as blood types or seed shapes. The insufficient amount of these types of characteristics in several organisms limited the mapping efforts that could be done.[1]

Some commonly used types of genetic markers are

• RFLP (or Restriction fragment length polymorphism)

• AFLP (or Amplified fragment length polymorphism

• RAPD (or Random amplification of polymorphic DNA)

• VNTR (or Variable number tandem repeat)

• Microsatellite polymorphism

• SNP (or Single nucleotide polymorphism)

• STR (or Short tandem repeat)

• SFP (or Single feature polymorphism)

• DArT (or Diversity Arrays Technology)

They can be further categorized as dominant or co-dominant. Dominant markers allow for analyzing many loci at one time, e.g. RAPD. A primer amplifying a dominant marker could amplify at many loci in one sample of DNA with one PCR reaction. Co-dominant markers analyze one locus at a time. A primer amplifying a co-dominant marker would yield one targeted product.

1. Restriction fragment length polymorphism

In molecular biology, the term restriction fragment length polymorphism, or RFLP, (commonly pronounced “rif-lip”) refers to a difference between two or more samples of homologous DNA molecules arising from differing locations of restriction sites, and to a related laboratory technique by which these segments can be distinguished. In RFLP analysis the DNA sample is broken into pieces (digested) by restriction enzymes and the resulting restriction fragments are separated according to their lengths by gel electrophoresis. Although now largely obsolete, RFLP analysis was the first DNA profiling technique cheap enough to see widespread application. In addition to genetic fingerprinting, RFLP was an important tool in genome mapping, localization of genes for genetic disorders, determination of risk for disease, and paternity testing.

Amplified fragment length polymorphism

Amplified Fragment Length Polymorphism PCR (or AFLP-PCR or just AFLP) is a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of genetic engineering. Developed in the early 1990’s by Keygene[1], AFLP uses restriction enzymes to cut genomic DNA, followed by ligation of adaptors to the sticky ends of the restriction fragments. A subset of the restriction fragments are then amplified using primers complementary to the adaptor and part of the restriction site fragments (as described in detail below). The amplified fragments are visualized on denaturing polyacrylamide gels either through autoradiography or fluorescence methodologies.

AFLP-PCR is a highly sensitive method for detecting polymorphisms in DNA. The technique was originally described by Vos and Zabeau in 1993[2][3]. In detail, the procedure of this technique is divided into three steps: [1]

1. Digestion of total cellular DNA with one or more restriction enzymes and ligation of restriction half-site specific adaptors to all restriction fragments.

2. Selective amplification of some of these fragments with two PCR primers that have corresponding adaptor and restriction site specific sequences.

3. Electrophoretic separation of amplicons on a gel matrix, followed by visualisation of the band pattern.

A variation on AFLP is cDNA-AFLP, which is used to quantify differences in gene expression levels.

Applications of AFLP:

The AFLP technology has the capability to detect various polymorphisms in different genomic regions simultaneously. It is also highly sensitive and reproducible. As a result, AFLP has become widely used for the identification of genetic variation in strains or closely related species of plants, fungi, animals, and bacteria. The AFLP technology has been used in criminal and paternity tests, in population genetics to determine slight differences within populations, and in linkage studies to generate maps for quantitative trait locus (QTL) analysis.

There are many advantages to AFLP when compared to other marker technologies including randomly amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), and microsatellites. AFLP not only has higher reproducibility, resolution, and sensitivity at the whole genome level compared to other techniques[4], but it also has the capability to amplify between 50 and 100 fragments at one time. In addition, no prior sequence information is needed for amplification (Meudt&Clarke 2007)[5]. As a result, AFLP has become extremely beneficial in the study of taxa including bacteria, fungi, and plants, where much is still unknown about the genomic makeup of various organisms.

RAPD:

RAPD (pronounced "rapid") stands for Random Amplification of Polymorphic DNA. It is a type of PCR reaction, but the segments of DNA that are amplified are random. The scientist performing RAPD creates several arbitrary, short primers (8-12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify. By resolving the resulting patterns, a semi-unique profile can be gleaned from a RAPD reaction.

No knowledge of the DNA sequence for the targeted gene is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where. This makes the method popular for comparing the DNA of biological systems that have not had the attention of the scientific community, or in a system in which relatively few DNA sequences are compared (it is not suitable for forming a DNA databank). Because it relies on a large, intact DNA template sequence, it has some limitations in the use of degraded DNA samples. Its resolving power is much lower than targeted, species specific DNA comparison methods, such as short tandem repeats. In recent years, RAPD has been used to characterize, and trace, the phylogeny of diverse plant and animal species