Molecular markers; the key to molecular breeding strategies
Authors: Prashant Raghunath Shingote, and Dipti Raghunath Dhumale

Progress in plant breeding has traditionally relied on the careful evaluation of the differences in physical characteristics or phenotypes between individual plants. Over the past 20 years, molecular marker technologies have been developed arising from the genetic differences between individual plants within a species. These marker technologies have been applied to plant breeding, enabling breeders to use the genetic composition or genotypes of plants as a criterion for selection and breeding progress. However, these methods have only been applied to a limited number of crop species, with the most widely used in rich countries. Even there, the application of molecular marker methods has been somewhat narrow in scope, focusing on a small number of traits or genomic regions.

Properties desirable for ideal DNA markers

i) Highly polymorphic nature

ii) Codominant inheritance (determination of homozygous and heterozygous states of diploid organisms)

iii) Frequent occurrence in genome

iv) Selective neutral behaviour

v) Easy access (availability)

vi) Easy and fast assay

vii) High reproducibility

viii) Easy exchange of data between laboratories.

Various types of molecular markers are utilized to evaluate DNA polymorphism and are generally classified ashybridization-based markers and polymerase chain reaction (PCR)-based markers.

Types and description of DNA markers

Restriction fragment length polymorphism (RFLP). RFLPs are simply inherited naturally occurring Mendelian characters. They have their origin in the DNA rearrangements that occur due to evolutionary processes, point mutations within the restriction enzyme recognition site sequences, insertions or deletions within the fragments, and unequal crossing over. It is a hybridization based marker. RFLP markers were used for the first time in the construction of genetic maps by Botstein et al., RFLPs, being codominant markers, can detect coupling phase of DNA molecules, as DNA fragments from all homologous chromosomes are detected. They are very reliable markers in linkage analysis and breeding and can easily determine if a linked trait is present in a homozygous or heterozygous state in individual, information highly desirable for recessive traits.

PCR based markers

Sequence-tagged sites (STS). RFLP probes specifically linked to a desired trait can be converted into PCR-based STS markers based on nucleotide sequence of the probe giving polymorphic band pattern, to obtain specific amplicon. Using this technique, tedious hybridization procedures involved in RFLP analysis can be overcome. This approach is extremely useful for studying the relationship between various species. When these markers are linked to some specific traits, 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.

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 was developed, wherein the denaturation step was 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 25.

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.

Direct amplification of minisatellite DNA markers (DAMD-PCR) . This technique, introduced by Heath et al., has been explored as a means of generating DNA probes useful for detecting polymorphism. DAMD-PCR clones can yield individual-specific DNA fingerprinting pattern and thus have the potential as markers for species differentiation and cultivar identification.

Inter simple sequence repeat markers (ISSR). These primers are 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 penta nucleotides [(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.

Randomly-amplified polymorphic DNA markers (RAPD). This procedure detects nucleotide sequence polymorphisms in DNA by using a single primer of arbitrary nucleotide sequence. 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 characterized amplified regions for amplification of specific band (SCAR) in this technique 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 markers48 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) These polymorphic patterns are generated by restriction enzyme digestion of PCR products. Such 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. These markers are codominant in nature.

Amplified fragment length polymorphism (AFLP) This 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.

AFLP analysis depicts unique fingerprints regardless of the origin and complexity of the genome. Most AFLP fragments correspond to unique positions on the genome and hence can be exploited as landmarks in genetic and physical mapping. AFLPs are extremely useful as tools for DNA fingerprinting58 and also for cloning and mapping of variety-specific genomic DNA sequences. Similar to RAPDs, the bands of interest obtained by AFLP can be converted into SCARs. Thus AFLP provides a newly developed, important tool for a variety of applications.

Applications of molecular markers in plant genome analysis and breeding. Molecular markers have been looked upon as tools for a large number of applications ranging from localization of a gene to improvement of plant varieties by marker-assisted selection. They have also become extremely popular markers for phylogenetic analysis adding new dimensions to the evolutionary theories. If we look at the history of the development of these markers, it is evident that they have been improved over the last two decades to provide easy, fast and automated assistance to scientists and breeders. Genome analysis based on molecular markers has generated a vast amount of information and a number of databases are being generated to preserve and popularize it. Mapping and tagging of genes: Generating tools for marker-assisted selection in plant breeding. 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. 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 program.

About Author / Additional Info:
I have completed my graduation and post graduation in Agricultural biotechnology. From last 4-5 years I have been working on different aspects of plant molecular genetics and functional genomics. I have published nearby 10 publication maximum of which are in peer reviewed journals having impact factors.