Molecular Approaches to Crop Improvement (2)

DNA Markers

It is estimated that each chromosome has 10⁸-10¹⁰ base pairs but only about 10% of the genome is actively coding with the result that a bulk of the genetic material remains unnoticed through phenotypic analysis. Furthermore, not much of the coded information may be easily observable that restricts the mapping of only a small fraction of the genome. The assessment of variation at the DNA level provides, at least in principle, mapping of each and every point of the entire genome. Specific spots on DNA molecule both in coding as well as non-coding regions can be identified as markers. In fact, the detection of naturally occurring DNA sequence polymorphism is the most attractive application of molecular biology for the welfare of mankind. A DNA marker is a small region of DNA showing sequence polymorphism in different individuals within a species or group of individuals. The interest in DNA based markers started from surprisingly highl level of variation for sequence  changes in DNA from different individuals.

Random Amplified Polymorphic DNAs (RAPDs)

This is a PCR based technique where a single short oligonucleotide primer which binds to many different loci, is used to amplify random sequences from a complex DNA template such as a plant genome. For most plants the primers that are 9-10 nucleotide long are expected to generate 2-10 amplification products (amplicon). The primers are generally of random sequence, biased to contain at least 50% GC content and to lack internal repeats. The products are easily separated by standard electrophoretic techniques and visualized by UV illumination of ethidium bromide stained gels. Pollymorphism results from changes in either the sequence of the primer binding site (e.g. point mutations) or from changes which alter the size or prevent the amplification of target DNA (e.g. insertions, deletions, inversions). In inheritance studies, the amplification products are transmitted as dominant markers (Waugh and Powell, 1992).

Molecular Approaches to Crop Improvement

MOLECULAR MARKERS

The progress in plant breeding is easy and quick for qualitative characters controlled by major genes with easily identifiable effect on the phenotype. But there are a number of plant characters which are difficult to be observed and where final phenotype does not give a reliable composition of the underlying genetic constitution. An individual may be carrying genes for resistance to say an insect but shall be known after it is exposed to insect attack. Similarly a male sterile plant cannot be identified before flowering stage and grain quality may not be known until before maturity of the crop. The identificatio of desirable genotypes for the quantitative characters like yield, quality, earliness, adaptation etc. is still more complicated. In all such cases the breeder attempts to select the desirable plants on the basis of some other easily observable attribute as a marker for the genes associated with the character of interest. Plant breeders have the convention of using many morphological characters as 'proxy' for the genes affecting complex characters. But the available number of such markers is too limited to serve as an index of each and every segment of DNA of the entire genome which may be controlling especially the quantitative characters. The available mutants can be assembled through breeding into the stocks under investigation but it requires additional efforts only for the purpose of finding association of genes with markers. The polymorphism at the DNA level provides a unique type of markers to serve as an index of genetic worth of the entire genome. An exact location of genes especially the loci governing quantitative traits (QTL) is basic and of paramount importance for map based cloning of these genes. Such markers can also be used to determine the nature of genetic variation in wild and cultivated types. But the most important use of these markers lies in the indirect manipulation of desirable genes in the form of marker-assisted selection (MAS). The following three classes of markers are available for use in plant breeding:

1. Morphological markers

2. Protein markers

3. DNA markers