-> to review and summarize scientific information on molecular marker techniques and their applications in the current plant breeding programs.
Specific objectives :
-> To differentiate the different molecular marker techniques.
-> To identify and describe the different molecular marker types.
-> To describe the different application of molecular markers.
-> To compare importance of molecular markers.
-> To describe limitation of molecular markers.
The three types of genetic markers:
-> traditional markers, highly dependent on environmental factors, time consuming, labour intensive; need large populations of plants and large plots of land or greenhouse space.
-> Proteins (Isozymes) or RNAs markers, less dependant on environmental factors, superior to morphological markers, less polymorphic differences (problem in commercial breeds of plants)
-> Enzymes are separated on the basis of net charge and mass via electrophoresis gels.
-> DNA marker, polymorphic, independent of environmental factors, easy, fast, co-dominant and reproducible but it is costly.
Molecular marker techniques
Two types of molecular techniques
1. Hybridization-based methods
-> The first, includes restriction fragment length polymorphism (RFLP) and variable number tandem repeats.
-> Fragments are separated using agarose or polyacrylamide gel electrophoresis.
-> Polymorphisms are detected by hybridizing the restriction enzyme-digested DNA with labelled probes.
-> Procedure for hybridization:
1) DNA isolation 2) Restriction digest
3) Gel electrophoresis
4) Transfer to nitrocellulose membrane filter
5) Probe visualization- probes consist of radio labeled sequences
2. Polymerase chain reaction (PCR)-based methods
-> Amplification of particular DNA sequences with arbitrary primers.
-> It includes random amplified polymorphic DNA (RAPD), and DNA amplification fingerprinting (DAF), Inter simple sequence repeats (ISSRs), amplified fragment length polymorphism (AFLP)
-> Amplified fragments are separated electrophoretically and banding patterns are detected by staining and autoradiography.
-> Procedure for PCR methods:
1) DNA fragmentaion with two restriction enzymes
2) Ligation of “adapters” to the ends of each fragment
3) Primers are designed from the known sequence of the adapter
4) PCR is performed
5) Visualization on agarose gels with ethidium bromide.
Table 1. Summary of advantages and disadvantages of some commonly used markers (Kumar et al. , 2009).
Table 2: Comparsion between different molecular methods for plant genome analysis (Sharma et al ., 2008).
Table 3. Comparison of the most common used markers (Kumar et al. , 2009).
Molecular marker applications in plant breeding
Molecular markers are used in plant breeding, taxonomy, physiology, embryology, genetics, evolution, genetic engineering etc
Some of applications in plant breeding are:
1. Marker-assisted selection
-> It replaces evaluation of a trait (difficult or expensive).
-> It is not influenced by growth stage and environmental factors.
-> It can increase efficiency of selection for low-heritability.
2. Genetic diversity analysis
-> Plant breeding is dependant of genetic variation
-> Genetic diversity is measured by genetic distance (GD) or genetic similarity (GS = 1 - GD)
-> It is used to assess variation over time, protection of intellectual property rights, registration of germplasm, and evaluation of new sources of germplasm for their potential to increase genetic diversity
-> It is superior to morphological, pedigree, heterosis, and biochemical
3. Genetic mapping
-> Markers can be used for map construction.
-> It predicts the linear arrangement of markers on a chromosome and the recombination frequency.
4. Manipulating traits controlled by a few major loci
-> To map and transfer the gene of interest
5. Authentication or DNA fingerprinting
-> For diagnosis, protection property right and quality controlling
6.Phylogeny and evolution
Perform phylogenic analysis on a species by comparing the presence/absence of various markers in their genome.
For taxonomic classification to determine the primary, secondary or tertiary genepool of the system
Plant breeding depends on genetic variation and selection.
Selection is difficult and assisted by morphological, biochemical and molecular markers.
Molecular markers are DNA marker, polymorphic, independent of environmental factors, easy, fast, co-dominant and reproducible but it is costly.
The two forms of molecular techniques( hybridization and PCR methods).
Many forms of molecular markers
-> RFLP, RAPD, VNTR, SSR, AFLP …….
Molecular markers are used in diversity analysis, parent selection, germplasm characterization, identification, genetic fingerprinting, genetic diagnostics, genome organization and phylogenic analysis