Genetic improvement of crop plant through conventional plant breeding has made tremendous contribution to the breakthrough in the global agricultural production. Recently, arrays of tools and techniques in the field of molecular biology have become available for supplementing the conventional genetic approaches. Consequently, new interacted approaches are being designed. One of the approaches employs molecular markers for genome mapping, gene tagging and marker assisted selection (MAS). With the complete sequencing of whole plant-genomes and a large number of random cDNAs in many different crops species, newer opportunities are emerging.
There are heritable differences in nucleotide sequences of DNA at corresponding position on homologous chromosomes of two different individuals, which follow a simple mendelin pattern of inheritance. These differences are detected employing mainly two basic techniques: (a) southern blot hybridization and (b) polymerase chain reaction (PCR). Briefly, southern hybridilization, developed by prof. EM southern in 1975 involves restriction of plant DNA electrophoretic sepration of DNA fragments, denatruation and transfer of fragments from gel to nylon membrane, hybridization of radiolabeled probe DNA and autoradiography to develop the X-ray films. The polymerase chain reaction, designed by Dr Kerry mullis in 1985. Involves denatruation to template DNA amplification, the fragments are separated on agarose or polyacrylamide gels obtain the DNA profiles. Using these two techniques and their variations, different "Molecular markers" or DNA markers' have been developed.
(i) Restriction fragment length polymorphism (RFLP),
(ii) Random amplified polymorphic DNA (RAPD),
(iii) Sequences tagged sites (STS),
(iv) Sequences tagged microsatellite sites (STMS)
(v) Amplified fragment length polymorphism (AFLP), and
(vi) single nucleotide polymorphism (SNP)
The RELP is based on Southern hybridization and the rest on polymerase chain reaction. These are briefly described below.
Restriction fragments length polymorphism (RFLP): RELP is the first molecular marker that was developed in 1980. It employees restriction enzymes that cut genomic DNA molecules at specific nucleotide sequences (restriction site), thereby yielding variable size DNA fragments. The genomic DNA fragments thus generated are separated by electrophoresis and transferred to nitrocellulose or nylon filter. The filter immobilized DNA is allowed to hybridize to n radioactively labeled probe DNA. Through X-ray autoradiography film, where radioisotope disintegration from the probe results in visible bands. Variation in the position of the bands revealing the difference in the size of the hybridizing augments is considered polymorphic. RFLP is a co-dominant marker in which the probes are usually small (500 to 3000bp), cloned genomic or cDNA fragments. RFLP markers are highly reproducible and thus very robust.
Sequence tagged sites (STS): This marker is highly class was first conceived and used in human genome mapping, and has been recently included in genome mapping in plants. STS markers are generated by an unmodified PCR. The primers are designed based on sequence of cDNA (expressed sequence tags, EST), random genomic DNA and ends of large genomic DNA fragment cloned in cosmid, lambda or artificial chromosomes. The STS marker system has all the advantages of PCR technique. STS markers are considered robust.
Random amplified polymorphic DNA (RAPD) : RAPD is a dominant marker based on PCR. It employs a single decamer primer of arbitrary sequence, which is annealed to the template DNA typically at 37%C. The variation in RAPD profile is in the form of presence or absence of band resulting from variation in primer binding sites. This marker system requires less amount of DNA per assay. Being based on random primers, it does not need prior knowledge of the sequence of the genomes. A large number of such primer are commercially available that work across organisms starting from bacteria to human. RAPD marker is technically less demanding and thus can be routine used in a small laboratory set by plant breeders/plant-protection specialists with a little exposure to molecular tools and techniques, A major limitation of this marker is non-reproducibility due to low annealing temperature.
Sequence tagged microsatellite sites (STMS): Simple sequence repeat (SSR) or microsatellites are tandem repeat of mono-,di-,tri-,tetra-and penta-nucleotide units dispersed throughout the genome. SSR polymorphism reflects variation in the number of repeat units in a defined region of the genome. Nucleotide sequence flanking the repeat is used to design primers to amplify different number of repeat units in different verities. These primers are very useful for rapid and accurate detection of polymorphic loci and the information could be used for developing a physical map based on these sequence tags. This type of polymorphism is highly reproducible .STMS markers are co dominant. These markers can be developed from the EST sequences and thus used to assay the expressed regions of the genome.
Amplified fragment length polymorphism (AFLP): The AFLP markers are generated by selective amplification of DNA fragments obtained by restriction enzyme digestion. High molecular weight DNA is digests by two restriction enzymes, one hexacutter (e.g. EcoR I) and one tetra cutter (e.g.Mse I). Adapter molecules are ligated to the end of DNA fragments. Two primers possessing sequence complementarily to the adapter as well as a few extra random nucleotides at their '3' ends are used for selective amplification of fragments employing PCR .The primer are labeled either by radioisotope or fluorescent dye so that the amplified products separated on sequencing gels/capillaries can be visualized by autoradiography or by laser based scanning in an automated fragments analysis system. Alternatively the products can be separated even using ordinary PAGE and visualized by silver staining. Maximum numbers of amplified products are obtained per reaction in AFLP among all the DNA profiling systems.
Single or simple nucleotide polymorphism (SNP): Polymorphism observed between individual/varieties may arise due to deletion/addition of multiple bases. Or due to single nucleotide substitution. The detection of such variation has led to the development of a few sequences based molecular marker called single/simple nucleotide polymorphism. SNPs are abundant in plant genomes. The SNP markers are being used for genotyping human populations for certain genetic diseases. Detection and assay of SNP has been automated that allows a large-scale genotyping in a far shorter period than the other markers.
Advantages of molecular markers
The molecular markers offer several advantages over the other genetic markers. These include: (i) abundance, (ii)co-dominance, (iii)phenotypic neutrality, (iv)absence of epistasis, and (V)developmental stage, tissue and environment independent expression.
Large number of molecular markers can be obtained either by using DNA probes from a variety of sources in combination with any of commercially available restriction enzymes or by using a variety of DNA primers. This enables study of several markers in a single population for construction of high-density linkage maps of plant genomes. The above attributes make molecular markers idea for characterization of quantitative traits.
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