PCR Based Markers: A great tool for Gene Tagging
Authors: Waghmare Deepak Harishchandra, Deshmukh Arti Madhukar

PCR (Polymerase Chain Reaction) based markers are of great interest to plant biotechnologist as a source of genetic information on crops and for use in indirect selection of traits to which the markers are linked. The use of molecular markers in plant breeding has become very commonplace and has given rise to “molecular breeding”. Molecular breeding involves primarily “gene tagging”, followed by “marker-assisted selection” of desired genes or genomes. Gene tagging refers to the identification of existing DNA or the introduction of new DNA that can function as a tag or label for the gene of interest. Various types of PCR based markers are utilized to evaluate DNA polymorphism and tag genes, such as Randomly-amplified polymorphic DNA markers (RAPD), Sequence-tagged sites (STS), Allele-specific associated primers (ASAPs), Expressed sequence tag markers (EST), Single strand conformation polymorphism (SSCP), Sequence-tagged microsatellite site markers (STMS), Inter simple sequence repeat markers (ISSR), Sequence characterized amplified regions (SCAR), Cleaved amplified polymorphic sequences (CAPs) and Amplified fragment length polymorphism (AFLP).

PCR-based markers involve in vitro amplification of particular DNA sequences or loci, with the help of specifically or arbitrarily chosen oligonucleotide sequences (primers) and a thermostable DNA polymerase enzyme. The amplified fragments are separated electrophoretically and banding patterns are detected. PCR is a versatile technique invented during the mid-1980s. Ever since thermostable DNA polymerase was introduced in 1988, the use of PCR in research and clinical laboratories has increased tremendously. The primer sequences are chosen to allow base-specific binding to the template in reverse orientation. PCR is extremely sensitive and operates at a very high speed. Its application for diverse purposes has opened up a multitude of new possibilities in the field of molecular biology.

Plant improvement, either by natural selection or through the efforts of breeders, has always relied upon creating, evaluating and selecting the right combination of alleles. The manipulation of a large number of genes is often required for improvement of even the simplest of characteristics. Evaluation of disease, herbicide, biotic and abiotic stress resistance, high yielding genotypes of crop plants by traditional method is time consuming, expensive and difficult. It is very much difficult to carry out disease resistance screening in sick plot. These markers offer a rapid evaluation of disease resistance genotypes in absence of pathogen. With the use of molecular markers it is now a routine to trace valuable alleles in a segregating population and mapping them. These markers once mapped enable dissection of the complex traits into component genetic units more precisely, thus providing breeders with new tools to manage these complex units more efficiently in a breeding programme.

Microsatellite markers, especially STMS markers, have been found to be extremely useful in this regard. Owing to their quality of following clear Mendelian inheritance, they can be easily used in the construction of index maps, which can provide an anchor or reference point for specific regions of the genome. Similar to microsatellites, looking at the pattern of variation, generated by retro-transposons, it is now proposed that apart from genetic variability, these markers are ideal for integrating genetic maps.

Once mapped, these markers are efficiently employed in tagging several individual traits that are extremely important for a breeding programme like yield, disease resistance, stress tolerance, seed quality, etc. A large number of monogenic and polygenic loci for various traits have been identified in a number of plants, which are currently being exploited by breeders and molecular biologists together, so as to make the dream of marker-assisted selection come true. Tagging of useful genes like the ones responsible for conferring resistance to plant pathogen, synthesis of plant hormones, drought tolerance and a variety of other important developmental pathway genes, is a major target. Such tagged genes can also be used for detecting the presence of useful genes in the new genotypes generated in a hybrid programme or by other methods like transformation, etc.

Randomly-amplified polymorphic DNA markers (RAPD)

This procedure detects nucleotide sequence polymorphisms in DNA by using a single primer of arbitrary nucleotide sequence. In this reaction, a single species of primer anneals to the genomic DNA at two different sites on complementary strands of DNA template. If these priming sites are within an amplifiable range of each other, a discrete DNA product is formed through thermocyclic amplification. On an average, each primer directs amplification of several discrete loci in the genome, making the assay useful for efficient screening of nucleotide sequence polymorphism between individuals. However, due to the stoichastic nature of DNA amplification with random sequence primers, it is important to optimize and maintain consistent reaction conditions for reproducible DNA amplification. They are dominant markers and hence have limitations in their use as markers for mapping, which can be overcome to some extent by selecting those markers that are linked in coupling. RAPD assay has been used by several groups as efficient tools for identification of markers linked to agronomically important traits, which are introgressed during the development of near isogenic lines. The application of RAPDs and their related modified markers in variability analysis and individual-specific genotyping has largely been carried out, but is less popular due to problems such as poor reproducibility, faint or fuzzy products, and difficulty in scoring bands, which lead to inappropriate inferences.

Sequence-tagged sites (STS)

STS markers based on nucleotide sequence of the probe giving polymorphic band pattern, to obtain specific amplicon. This approach is extremely useful for studying the relationship between various species. When these markers are linked to some specific traits, for example powdery mildew resistance gene or stem rust resistance gene in barley, they can be easily integrated into plant breeding programmes for marker-assisted selection of the trait of interest.

Allele-specific associated primers (ASAPs)

To obtain an allele-specific marker, specific allele (either in homozygous or heterozygous state) is sequenced and specific primers are designed for amplification of DNA template to generate a single fragment at stringent annealing temperatures. These markers tag specific alleles in the genome and are more or less similar to SCARs.

Expressed sequence tag markers (EST)

These markers are obtained by partial sequencing of random cDNA clones. Once generated, they are useful in cloning specific genes of interest and synteny mapping of functional genes in various related organisms. ESTs are popularly used in full genome sequencing and mapping programmes underway for a number of organisms and for identifying active genes thus helping in identification of diagnostic markers. Moreover, an EST that appears to be unique helps to isolate new genes.

Single strand conformation polymorphism (SSCP)

This is a powerful and rapid technique for gene analysis particularly for detection of point mutations and typing of DNA polymorphism. SSCP can identify heterozygosity of DNA fragments of the same molecular weight and can even detect changes of a few nucleotide bases as the mobility of the single-stranded DNA changes with change in its GC content due to its conformational change. To overcome problems of reannealing and complex banding patterns, an improved technique called asymmetric-PCR SSCP is used, wherein the denaturation step eliminated and a large-sized sample could be loaded for gel electrophoresis, making it a potential tool for high throughput DNA polymorphism. It was found useful in the detection of heritable human diseases. In plants, however, it is not well developed although its application in discriminating progenies can be exploited, once suitable primers are designed for agronomically important traits.

Sequence-tagged microsatellite site markers (STMS)

This method includes DNA polymorphism using specific primers designed from the sequence data of a specific locus. Primers complementary to the flanking regions of the simple sequence repeat loci yield highly polymorphic amplification products. Polymorphisms appear because of variation in the number of tandem repeats (VNTR loci) in a given repeat motif. Tri- and tetranucleotide microsatellites are more popular for STMS analysis because they present a clear banding pattern after PCR and gel electrophoresis. However, dinucleotides are generally abundant in genomes and have been used as markers e.g. (CA) n (AG)n and (AT)n. The di- and tetranucleotide repeats are present mostly in the non-coding regions of the genome, while 57% of trinucleotide repeats are shown to reside in or around the genes. A very good relationship between the number of alleles detected and the total number of simple repeats within the targeted microsatellite DNA has been observed. Thus larger the repeat number in the microsatellite DNA, greater is the number of alleles detected in a large population.

Inter simple sequence repeat markers (ISSR)

In this technique, primers based on microsatellites are utilized to amplify inter-SSR DNA sequences. Here, various microsatellites anchored at the 3’ end are used for amplifying genomic DNA which increases their specificity. These are mostly dominant markers, though occasionally a few of them exhibit codominance. An unlimited number of primers can be synthesized for various combinations of di-, tri-, tetra- and pentanucleotides [(4)3 = 64, (4)4 = 256] etc. with an anchor made up of a few bases and can be exploited for a broad range of applications in plant species.

Sequence characterized amplified regions (SCAR)

In this technique the RAPD marker termini are sequenced and longer primers are designed (22–24 nucleotide bases long) for specific amplification of a particular locus. These are similar to STS markers in construction and application. The presence or absence of the band indicates variation in sequence. These are better reproducible than RAPDs. SCARs are usually dominant markers, however, some of them can be converted into codominant markers by digesting them with tetra cutting restriction enzymes and polymorphism can be deduced by either denaturing gel electrophoresis or SSCP. Compared to arbitrary primers, SCARs exhibit several advantages in mapping studies (codominant SCARs are informative for genetic mapping than dominant RAPDs), map-based cloning as they can be used to screen pooled genomic libraries by PCR, physical mapping, locus specificity, etc. SCARs also allow comparative mapping or homology studies among related species, thus making it an extremely adaptable concept in the near future.

Cleaved amplified polymorphic sequences (CAPs)

Most Cleaved amplified polymorphic sequences markers are codominant and locus-specific in nature. Polymorphisms are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites in PCR amplicons produced by locus-specific primers. The digests are compared for their differential migration during electrophoresis. PCR primer for this process can be synthesized based on the sequence information available in databank of genomic or cDNA sequences or cloned RAPD bands.

Amplified fragment length polymorphism (AFLP)

It is a technique based on the detection of genomic restriction fragments by PCR amplification and can be used for DNAs of any origin or complexity. The fingerprints are produced, without any prior knowledge of sequence, using a limited set of generic primers. The number of fragments detected in a single reaction can be ‘tuned’ by selection of specific primer sets. AFLP technique is reliable since stringent reaction conditions are used for primer annealing. This technique thus shows an ingenious combination of RFLP and PCR techniques and is extremely useful in detection of polymorphism between closely related genotypes.

About Author / Additional Info:
I am currently working as junior research fellow on Department of biotechnology, new delhi, funded project "Gene tagging for fusarium wilt resistance in safflower" at VDCOAB, Latur, Vasantrao Naik Marathwada Agricultural University, Parbhani