A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify cells, individuals or species. It can be described as a variation which may arise either due to genetic effect (mutation or alteration in the genomic loci) or environmental effect and may represent polymorphism (genetic differences) between individuals of same or different species, in a population or between populations in a species. Generally, they do not represent the target genes themselves but act as 'signs' or 'flags'. They are located in close proximity to genes (i.e. tightly linked) may be referred to as gene 'tags'. Such markers themselves do not affect the phenotype of the trait of interest because they are located only near or 'linked' to genes controlling the trait. Like genes, all the genetic markers occupy specific genomic positions within the chromosomes and are associated with the particular trait.Such genetic maps serve several puposes, including detailed analysis of associations between economically important traits and genes or quantitative trait loci (QTLs)and facilitate the introgression of desirable genes or QTLs through marker assisted seolection. Genetic markers as well as linkage maps were described before the discovery of proteins and DNA.
Genetic markers can be divided into three main categories:
a. Morphological markers: visually accessible traits such as flower color, seed shape, growth habits or pigmentation
b. Biochemical markers: allelic variants of enzymes called isozymes that are detected by electrophoresis and specific staining. They are able to detect diversity at functional gene level and have simple inheritance
c. Molecular markers: based on DNA assays. Such markers themselves do not affect the phenotype of the trait of interest because they are located only near or 'linked' to genes controlling the trait.
All genetic markers occupy specific genomic positions within chromosomes (like genes) called loci. The major disadvantages of morphological and biochemical markers are that they may be limited in number and are influenced by environmental factors or the developmental stage of the plant. However, despite these limitations, morphological and biochemical markers have been extremely useful to plant breeders. DNA markers are the most widely used type of marker predominantly due to their abundance. They arise from different classes of DNA mutations such as substitution mutations (point mutations), rearrangements (insertions or deletions) or errors in replication of tandemly repeated DNA. These markers are selectively neutral because they are usually located in non-coding regions of DNA. Unlike morphological and biochemical markers, DNA markers are practically unlimited in number and are not affected by environmental factors and/or the developmental stage of the plant.
Classification of molecular markers:
Molecular markers reveal genetic differences that can be visualized by using a technique called gel electrophoresis and staining with chemicals (ethidium bromide or silver) or detection with radioactive probes. Markers that reveal differences between any two individuals are called polymorphic markers whereas that does not discriminate between genotypes are called monomorphic markers. Polymorphic markers may also be described as co-dominant or dominant. This description is based on whether markers can discriminate between homozygotes and heterozygotes. Co-dominant markers indicate differences in size, can analyze one locus at a time whereas dominant markers indicate difference based on their presence or absence and can analyze many loci at one time.
Different forms of a DNA marker (i.e. different sized bands on gels) are called marker alleles. Co-dominant markers may have many different alleles whereas a dominant marker only has two alleles.Ideal marker system should have these properties:
1. Easily available and identifiable
2. Associated with a specific locus
3. Easy and rapid assay
4. Highly variable, polymorphic and reproducible
5. Co-dominant inheritance and recurrent occurrence in genome
6. Selectively neutral to environmental conditions or management practices
7. Easy data exchange between different laboratories
8. Cost and time effective
DNA markers may be broadly divided into three classes based on the method of their detection:
(1) Hybridization based
(2) Polymerase chain reaction based and
(3) DNA sequence-based.
Essentially, DNA markers may reveal genetic differences that can be visualised by using a technique called gel electrophoresis and staining with chemicals (ethidium bromide or silver) or detection with radioactive or colorimetric probes. DNA markers are particularly useful if they reveal differences between individuals of the same or different species.
1) Hybridization based markers
In hybridization based markers, DNA profiles are visualized by hybridizing the restriction endonuclease digested DNA fragment, to a labeled probe, which is a DNA fragment of known sequence e.g. Restriction fragment length polymorphism (RFLP). RFLPs are single or low copy DNA fragments and are simply inherited. These probes could be genomic clones, cDNA clones or even cloned genes. They show co-dominance and are highly reliable in linkage analysis and breeding. Their detection is based on radio-labelling, require large amounts of DNA, are labour intensive and relatively expensive and hazardous and only one marker may be polymorphic which is highly inconvenient especially for crosses between closely related species and their inability to detect point mutations and polymorphism. Hence their large scale use in practical plant breeding may be restricted. They have their DNA rearrangements due to evolutionary processes, point mutations within the restriction enzyme recognition site, mutations within the fragments and unequal crossing over.
2) PCR based markers
PCR based markers involve in vitro amplification of particular DNA sequence with the help of specifically or arbitrarily chosen oligonucleotide sequences (primers) and a thermostable DNA polymerase enzyme. The amplified DNA fragments are separated by electrophoresis and banding patterns are detected by different methods such as staining (using ethidium bromide dye) and autoradiography.
i. Sequence tagged sites (STS): The RFLP probes, linked to desirable traits can be converted to PCR based markers. In this, the RFLP probes are end-sequenced and complementary primers are synthesized. These primers generally (20mers) are then used for amplifying specific genomic sequences using PCR
ii. Expressed sequence tags (EST): These markers are developed by end sequencing of random cDNA clones. The cDNA markers are first mapped as RFLP markers and then partially sequenced to convert them into PCR based markers. Thus, these are like STS markers. These can be used for synteny mapping and cloning of specific genes.
iii. Simple sequence repeats (SSR): This term was coined by Litt et al in 1989. Also called the microsatellites, originally designated as short tandem repeats (STRs) dispersed throughout the genome. These are generally di- to tetra nucleotide repeats and are highly hypervariable and thus highly polymorphic. These are flanked with unique sequences that are highly conserved. The flanking unique sequences are analyzed and their complementary primers synthesized. These can thus be assayed with PCR (Weber and May 1989) and act as co-dominant markers. Thence, they can easily differentiate between heterozygotes and homozygotes. These are also referred to as simple sequence length polymorphism (SSLP). Allelic differences are usually a result of variable number of repeat units (VNTRs) or hypervariable regions (HVRs).
iv. Random amplified polymorphic DNA (RAPD): Williams et al (1990) originally developed the technique. In this arbitrary decamer sequences are used as primers for amplification. These markers are dominant markers because the polymorphism is due to presence or absence of a particular amplified fragment. One major advantage of these markers is that this does not need any prior sequence informatio. One major limitation is lack of repeatability in certain cases.
v. Sequence characterized amplified regions (SCAR): This technique was introduced by Michelmore et al (1991) and Martin et al (1991). These markers overcome the limitations of RAPDs. In this, the RAPD fragments that are linked to a gene of interest are cloned and their termini sequenced. Based on the terminal sequences, longer primers (20-mers) are designed. These SCAR primers lead to a more 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 sequences. The SCAR markers thus are dominant markers.
vi. Amplified fragment length polymorphism (AFLP): In this technique, restriction fragments generated by a frequent (4 base) and a rare (6 base) cutter are anchored with oligo-nucleotide adapters of new oligo-nucleotide bases. This method generates a large number of restriction fragments facilitating the detection of polymorphism. The number of DNA fragments amplified can be controlled, by choosing different base numbers and composition of nucleotides in the adapters. This technique is more reliable since stringent reaction conditions are used for primer annealing. This technique thus shows a combination of RFLP and PCR technique and is extremely useful in detection of polymorphism between closely related genotypes.
vii. Diversity array technology (DArT): Diversity array technology is a high throughput DNA polymorphism analysis method that combines miniaturization of microarrays and restriction based PCR. It is capable of providing comprehensive genome coverage even in organisms without any DNA sequence information. It is a solid state open platform method for analyzing DNA polymorphism. This is a high throughput relatively low cost technique. DArT has been used in whole genome sequence processing of barley and for high throughput profiling of hexaploid wheat genome.
viii. Cleaved amplified polymorphic sequences (CAPS): These polymorphic patterns are generated by restriction enzyme digestion of PCR products. Such products are compared for their differential migration during electrophoresis. PCR primers for this process can be synthesized based on the sequence information available in the database of genomic or cDNA sequence or cloned RAPD bands. These markers are co-dominant in nature.
3) DNA sequence based markers
i. Single nucleotide polymorphism (SNP): Compared to the gel based molecular marker system, SNP determination and analysis can be carried out with non-gel based assays. SNPs are DNA sequence variations that occur when a single nucleotide (A, T, C or G) in a genome sequence is changed. Most SNPs (actually 2 out of every 3 SNPs) involve the replacement of cytosine (C) with thymine (T). SNPs occurs every 100 to 300 bases along the genome. Two types of nucleotide base substitution resulting in SNPs are transition (occurs between purines-A and G) or pyrimidines (C and T) and transversion (occurs between purine and pyrimidine). Transition type of substitution constitutes 2/3rd of all SNPs and successful amplification is detected by fluorescent dyes and is allele specific. They can be generated by sequencing, single strand conformational polymorphism, aligning and comparing multiple sequences of the same region from public genome and EST databases. They can be genotyped either by allele specific hybrization, primer extension, oligo-nuclotide ligation or invasive cleavage. Its advantages include, if the exact nature and location of the allelic variation is known, large number of samples can be screened for a marker using a variety of high throughput techniques. These are likely to occur at higher frequencies in less conserved genes.
Table 1: Comparative study of different markers
| Isozyme | RFLP | RAPD | AFLP | SSR | SNP | |
| Abundance | Low | Medium | Very high | Very high | High | Very high |
| Types of polymorphism | Amino acid change in polypeptide | Single base change, insertion, deletion, inversion | Single base change, insertion, deletion, inversion | Single base change, insertion, deletion, inversion | Repeat length variation | Single base change |
| DNA quality | - | High | Medium | High | Medium | Medium |
| DNA sequence information | - | Not required | Not required | Not required | Required | Required |
| Level of polymorphism | Low | Medium | High | High | High | High |
| Inheritance | Co-dominance | Co-dominance | Dominance | Dominance | Co-dominance | Co-dominance |
| Reproducibility | Medium | High | Low | Medium | High | High |
| Technical complexity | Medium | High | Low | Medium | Low | Medium |
| Developmental cost | Medium | High | Low | Low | High | High |
| Automation | Low | Low | Medium | Medium | High | High |
Application of genetic markers
Genetic markers can be used for crop improvement in several ways:
1. DNA fingerprinting for varietal identification
Inorder to protect varieties developed by originating plant breeder Plant Breeder's rights have been provided. IN order to achieve this, DNA fingerprinting can be used for varietal identification as well as for ascertaining variability in the germplasm. RAPDs, microsatellites and AFLPs are the markers of choice for the DNA fingerprinting. The fingerprinting information is useful for quantification of genetic diversity, characterization of accessions in plant germplasm collections and for protection of germplasm especially the CMS lines. These markers have been used to differentiate even closely related cultivars.
2. Germplasm evaluation
Evaluation of germplasm is important for differentiating cultivars, for construction of heterotic groups, for identification of germplasm redundancy, for monitoring genetic shifts that occur during germplasm storage, regeneration, domestication and breeding, for screening germpalsm for novel/superior genes (alleles) and for constructing a representative subset or core collection. SSRs are suitable for purity control and differentiation of plant cultivars.
3. Phylogenetic and evolutionary studies
To discern evolutionary relationships within and between species, genera or larger taxonomic groupings genetic markers are of use. Such studies involve studying similarities and differences among taxa using numerous genetic markers (Paterson et al 1991). Although phylogenetic trees have previously been established for many species on the basis of visible and isozyme markers and chromosome homology, the DNA markers have recently added to length and (breadth to phylogenetic information available for a number of species. RAPDs in wheat, ISSRs in rice and RFLPs have been used more frequently for phylogenetic and evolutionary studies. RFLPs have been used for assuming the relationship between hexaploid genome of bread wheat and its ancestors.
4. Development of saturated maps
Saturated linkage maps are a pre-requisite for gene tagging, marker assisted selection and map based gene cloning. Saturated linkage maps have been developed in several crop plants like maize, rice, tomato, wheat, potato, barley, cotton, Brassica etc. In rice centromere positions in all the 12 linkage groups have been defined. The markers used for developing saturated maps include RFLPs, RAPDs, SSRs, AFLPs, SNPs, DArT and a combination of these.
5. Gene tagging
Gene tagging refers to mapping of genes of economic importance close to known markers so that gene and marker cosegregates. With the construction of molecular map, especially the RFLP maps, several genes of economic importance like disease resistance (xa 1,2,3,4,5,8,13,21,26,27,29,30 in rice), stress tolerance, insect resistance, fertility restoration genes (Rfg1 in rye), yield attributing traits etc. have been tagged.
6. Marker assisted selection
Marker assisted selection (MAS) is useful for gene pyramiding, marker assisted alien introgression and simultaneous identification and pyramiding of QTLs from primitive cultivars and alien species. Jena et al (1992) used RFLP markers for detecting introgression in interspecific backcross derived lines of the cross O. sativa (AA) x O. officinalis (CC). QTLs for yield and yield improving traits have been identified and transferred into rice O. sativa from its wild relative O. rufipogon. MAS has been utilized for incorporation of xa 13 and Xa 21 of bacterial nlight resistance in rice and two varieties have been released in India Samba Mahsuri and Pusa Basmati 1 for cultivation.
7. Comparative mapping
Molecular markers are being used extensively for studying the divergence and evolution of crop plants. In this marker clones especially cDNA clones of one crop plant are being mapped onto the linkage maps of other crops. This approach of comparative mapping is useful in several ways:
i. More saturated maps can be generated by mapping marker clones of one crop onto the linkage map of other crops.
ii. By cross mapping, divergence and evolutionary history of various crop plants can be revealed.
iii. It can make gene cloning from complex organisms comparatively easier.
The first comparative maps in plants were generated in tomato, pepper and potato genomes by Tanksley et al 1988. Since then comparative maps have been generated in several crops especially in grasses, rice and wheat.
8. Map-based gene cloning
It refers to the isolation of a gene corresponding to a target trait using molecular maps. Map-based cloning consists of four major steps:
i. Development of a high-resolution molecular linkage maps in the region of interest.
ii. Physical mapping of the region of interest by yeast artificial chromosome (YAC) or bacterial artificial chromosome (BAC) contigs.
iii. Identification of appropriate YAC or BAC clones for isolating putative clones harbouring the gene of interest.
iv. Verification through transformation that the target gene is isolated.
Martin et al (1992) was the first to clone a disease resistance gene Pto in tomato using map-based cloning.
9. Molecular markers in heterosis breeding
One of the earliest uses of molecular markers was its use in heterosis breeding. Lee et al (1989) suggested that RFLP analysis might provide an alternative to field-testing when attempting to assign maize inbred lines to heterotic groups. Melchinger et al (1991) analyzed 32 maize inbred lines for molecular marker diversity. Zhang (1995) on the other hand observed a high correlation between specific heterozygosity and mid parent heterosis in rice. Stuber et al (1992) and Bains et al (1999) mapped QTLs contributing to heterosis in the cross between the elite maize inbred lines B73 and Mo17. Xiao et al (1995) mapped QTLs for heterosis in one of the highest yielding indica x japonica hybrids.
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