Molecular Markers and their Application in Plant Improvement | Agriculture Optional Notes for UPSC PDF Download

What is a Marker?

• A marker refers to a variation or difference in alleles at a specific DNA locus, which can be observed at various levels such as morphological, biochemical, or molecular.
• Molecular markers are based on natural changes or polymorphisms in the DNA sequence, which can involve deletions, substitutions, additions, tandem repeats, or duplications.
• All molecular markers have specific positions within the chromosome, known as "loci."
• Markers that are closely situated to desirable genes are referred to as "gene tags."
• Traits of agricultural significance are often influenced by multiple genes, a condition termed "polygenic" for quantitative traits.
• Within the genome, there are regions containing genes associated with a particular quantitative trait, and these are called "Quantitative Trait Loci" or QTLs.

Why Use Molecular Markers?

• Molecular markers are preferred because they are selectively neutral, located in the non-coding regions of the genome, and thus do not influence the functioning of genes directly.
• These markers are co-inherited along with the specific trait of interest, making them valuable for tracking and identifying desired characteristics.
• Molecular markers strictly adhere to the Mendelian pattern of inheritance, providing a reliable and predictable means of genetic analysis.
• They are free from complicating factors like epistatic interactions or pleiotropic effects, simplifying the study of individual traits.

Characteristics of an Ideal Molecular Marker

• It should display polymorphism, meaning it has variable forms among individuals.
• The marker should be co-dominant, so both alleles are distinguishable.
• It should be reproducible, consistently yielding the same results.
• The marker needs to be robust, capable of withstanding various conditions.
• Cost-effectiveness is crucial to make it practical for widespread use.
• Ease of use is important, allowing for straightforward application.
• High throughput capacity for processing a large number of samples efficiently.
• The marker should be closely linked to the trait of interest.

Levels of Marker Analysis

• Morphological markers are based on visual or phenotypic plant characteristics.
• Biochemical markers are related to gene product, often categorized as protein-based or isozyme-based.
• DNA markers, or genetic markers, involve the analysis of DNA sequences.

1. Morphological Markers:

• These markers are botanical descriptors of plants characterized by their visual or phenotypic traits.
• They are also known as DUS (Distinctiveness, Uniformity, Stability) descriptors or universal markers.
• Examples include seed color, seed shape, seed size, flower color, growth habits, and plant pigmentation.

2. Biochemical Markers:

• These markers come in two types: protein-based and enzyme-based (isozyme).
• The first true biochemical marker was an allelic variant of the enzyme pyruvate dehydrogenase.
• Isoforms can be separated by gel electrophoresis based on size, shape, and amino acid differences.
• They are easily assayed and detected but tend to exhibit lower polymorphism compared to DNA markers.

4. DNA-based Markers:

• These markers can be classified based on their ability to distinguish between homozygotes and heterozygotes.
• Co-dominant markers can discriminate between both types, while dominant markers do not.
• They can be visualized through gel electrophoresis, ethidium bromide or silver staining, and the use of radioactive or colorimetric probes.

Comparison between Co-dominant and Dominant Markers

• Co-dominant markers distinguish between homozygotes and heterozygotes.
• Dominant markers do not differentiate between these genetic states.

Classification of Molecular Markers

• Hybridization-based markers include various types such as RAPD, RFLP, AFLP, SSR, ISSR, SNP, SCAR, CAPS, TRAP, DArT, DAF, and SRAP.

Full Forms of Some Markers

• RAPD: Random Amplified Polymorphic DNA
• RFLP: Restriction Fragment Length Polymorphism
• AFLP: Amplified Fragment Length Polymorphism
• SSR: Simple Sequence Repeat
• ISSR: Inter Simple Sequence Repeat
• SNP: Single Nucleotide Polymorphisms
• SCAR: Sequence Characterized Amplified Region
• CAPS: Cleaved Amplified Polymorphic Sequence
• TRAP: Target Region Amplification Polymorphism
• DArT: Diversity Arrays Technology
• DAF: DNA Amplification Fingerprinting
• SRAP: Sequence Related Amplified Polymorphism Markers

Classification Based on Principles and Detection Methods

A. Hybridization-Based Markers:

• RFLP (Restriction Fragment Length Polymorphism):
  • Arise from polymorphism due to chromosomal aberrations in specific DNA regions.
  • Co-dominant, allowing for the identification of unique loci.
  • Utilizes known-function DNA probes.
  • Restriction enzymes recognize and cut DNA at specific short sequences.
  • Molecular probes used for Southern hybridization may be radioactive or non-radioactive.
  • Fragment sizes can be compared through electrophoresis.

B. PCR-Based Markers:

• Streamline time, effort, and expenses.
• Relies on a pair of primers (reverse and forward).
• Primers can be designed based on random sequences or specific sequences flanking the DNA segment to be amplified.

Two subtypes:

• Single primer used as both forward and reverse primer (e.g., AP-PCR, RAPD, DAF).
• A pair of primers used (e.g., STSs, SCARs, STARs).

About Primers

Primers are short DNA sequences, approximately 20 base pairs long, with a free 3'-OH group. They are typically used to amplify marker loci.

i. RAPD (Random Amplified Polymorphic DNA):

• The first PCR-based marker technique, known for its simplicity.
• Uses short PCR primers (around 10 base pairs) selected at random to amplify DNA segments throughout the genome.
• Results in amplification products generated at regions flanking a part of the 10 base pair priming sites.
• RAPD is a dominant marker and is visualized on agarose gels stained with ethidium bromide.
• A modified approach of RAPD is DNA Amplification Fingerprinting (DAF).

ii. AFLP (Amplified Fragment Length Polymorphism):

• Differences in restriction fragment lengths arise from SNPs and INDELs that create or abolish restriction sites.
• Based on selective PCR amplification of restriction fragments from total genomic DNA digestion.
• Involves the ligation of oligonucleotide adapters to the ends of DNA fragments, followed by PCR amplification.
• SCARs (Sequence Characterized Amplified Region):
  • Based on sequences of polymorphic bands from RAPD, RFLP, or AFLP linked to the trait of interest.
  • Uses longer primers (15-30 base pairs) designed for the specific amplification of a particular locus.
  • Demonstrates higher reproducibility compared to RAPD and RFLP.
  • Co-dominant in nature.

iii. Single Nucleotide Polymorphism (SNPs):

• SNP denotes DNA sequence variations occurring when a single nucleotide (A, T, C, or G) in the genome differs between individuals within a species.
• Can only be known after DNA sequencing of multiple genomes.
• Applied in biomedical research and crop and livestock breeding programs.

iv. Simple Sequence Repeats (SSRs):

• Also referred to as Short Tandem Repeats (STRs) or microsatellites, which are repeating sequences of 2-6 nucleotides of DNA (motifs).
• Co-dominant markers that are abundant and dispersed throughout the genome.
• Occur in DNA when two or more nucleotides are repeated in patterns adjacent to each other.
• Types include EST-SSRs, Genomic-SSRs, Mitochondrial-SSRs, and Chloroplastic-SSRs.
• Expressed Sequence Tags (ESTs) represent transcribed regions in a DNA sequence from a cDNA clone corresponding to mRNA, making them highly transferable and valuable in developing STSs, SSRs, and SNPs.
• Widely utilized in the Rice genome.

Applications of Molecular Markers

• Confirmation of hybridity.
• Linkage mapping.
• Marker-assisted selection (MAS).
• Trait-based selection.
• Saturated maps.
• Orthologous gene mapping.
• Gene tagging.
• Heterosis breeding.
• Haplotype mapping.
• DNA fingerprinting for varietal identification.
• Phylogenetic and evolutionary studies.

Confirmation of Hybridity

• Detecting heterozygosity in F1 hybrids.

Linkage Mapping

• Requires mapping populations that are immortal, universal, homozygous (true breeding type), and do not fluctuate.
• Utilizes various populations such as BC1F2, F2, DH, F2:F3, RILs, NILs.

Marker Assisted Selection (MAS)

• Involves identifying associations between molecular markers and genes controlling agronomic traits (major genes).
• Selection is based on genotype rather than phenotype, leading to faster and more efficient selection.
• Used for manipulating both qualitative (e.g., disease resistance) and quantitative (e.g., yield) traits.
• Molecular markers increase the probability of identifying superior genotypes by eliminating inferior ones early.

X Back Crossings

• Plant breeders improve the resistance of a cultivated species by crossing it with a wild species possessing the desired resistance.
• Typically, at least six backcrossing steps are necessary to incorporate 99% of the genome from the recurrent parent.

A Robust Marker in Disease Resistance

• A robust marker is developed for a major QTL controlling disease resistance, enabling plant breeders to replace large field trials and eliminate undesirable plants (up to 75%).

Trait-Based Selection

• Molecular markers have been used for indirect selection of genes in breeding programs.
• In South Australian Barley, a single dominant resistance gene to cereal cyst nematode (Ha2) was transferred from a resistant to a susceptible variety using three cycles of marker-assisted backcrossing with a single molecular marker.

Orthologous Gene Mapping

• Also known as "Comparative mapping."
• Maps cDNA clones of one crop plant onto the linkage maps of other crops.
• Maps can be made more saturated by mapping marker clones.

Gene Tagging

• Refers to mapping genes of agricultural importance located close to known markers.
• A molecular marker closely linked to a gene can serve as a "tag."
• Gene tagging is a prerequisite for MAS and map-based gene cloning.
• Examples include tagging cereal cyst nematode genes in wheat and tagging leaf rust resistance genes in Brassica.

Heterosis Breeding

• Molecular markers have been used to correlate heterosis with variability at the molecular level.
• Various studies have mapped QTLs contributing to heterosis in different crops, demonstrating the significance of dominance in heterosis.

DNA Fingerprinting for Varietal Identification

• DNA fingerprinting using markers like RAPD, microsatellite, and AFLPs is essential for identifying crop varieties.
• Particularly useful for protecting proprietary germplasm, characterizing germplasm collections, and sex identification in dioecious plants.

Phylogenetic and Evolutionary Studies

• Molecular markers are employed to study evolutionary relationships within and between species, genera, or larger taxonomic groups.
• These studies analyze genetic markers such as RAPD and RFLP to determine similarities and differences among taxa, aiding in the classification of species, like the example involving rice lines Azueena and PR 304.