Unit 10: PCR & Molecular markers

Molecular markers are heritable, identifiable DNA sequences whose variation between individuals is detected in the laboratory to fingerprint, map and classify plant genomes. This unit couples the polymerase chain reaction (PCR)—the technique that amplifies target DNA to detectable amounts—with the marker systems that read genetic polymorphism from a given plant DNA sample. The single practical objective is the molecular analysis of extracted plant DNA using DNA-based markers, from template preparation through amplification, electrophoresis and band scoring.

Defining features that later sections rely on:

• Template: high-molecular-weight genomic DNA isolated from plant tissue (typically by CTAB extraction), free of polysaccharides, polyphenols and nucleases that inhibit Taq polymerase.
• Polymorphism: the observable difference in marker profile (band presence/absence, fragment length, or single-base identity) that distinguishes genotypes.
• Marker classes: hybridisation-based (RFLP) versus PCR-based (RAPD, AFLP, SSR, ISSR, SNP).
• Inheritance: markers are dominant (band present/absent, cannot separate heterozygotes) or codominant (both alleles visible, heterozygotes distinguishable).
• Readout: agarose or polyacrylamide gel electrophoresis, resolving fragments by size against a DNA ladder.

II. Polymerase Chain Reaction — the amplification engine

A. Principle and components

PCR exponentially copies a defined DNA segment through repeated enzymatic replication in vitro.

• Template DNA: the plant genomic DNA carrying the region to be amplified.
• Primers: two short oligonucleotides (18–25 nt) flanking the target; a forward primer binds the antisense strand, a reverse primer the sense strand.
• dNTPs: dATP, dTTP, dGTP, dCTP supplying nucleotide building blocks (~200 µM each).
• DNA polymerase: thermostable Taq from Thermus aquaticus, active near 72 °C.
• Buffer + Mg²⁺: MgCl₂ (1.5–2.5 mM) is an essential cofactor; excess causes non-specific bands, too little lowers yield.

Amplification is exponential: copies ≈ initial × 2ⁿ, where n is the cycle number, so 30 cycles theoretically yield ~10⁹-fold amplification.

B. Thermal cycling steps

Each cycle repeats three temperature-controlled stages.

1. Denaturation   94–95 °C, 30 s   → strands separate
2. Annealing      50–65 °C, 30 s   → primers bind template
3. Extension      72 °C, 1 min/kb  → Taq synthesises new strand

• Annealing temperature (Ta): set ~5 °C below primer melting temperature. A quick estimate:

Tm = 4(G + C) + 2(A + T)

where G, C, A, T are counts of each base in the primer. High GC primers need higher Ta.

• Final extension: 72 °C for 5–10 min completes partial products.

C. Variants used in marker work

• Standard PCR: single primer pair for a known locus (used in SSR, SNP assays).
• Arbitrary-primer PCR: a single random primer amplifies anonymous loci (basis of RAPD).
• Selective PCR: adaptor-primed amplification with selective bases (basis of AFLP).

III. DNA-based molecular markers

A. Definition and desirable properties

A DNA marker is a locus whose allelic variation is scored directly at the DNA level, independent of the plant's phenotype or growth stage.

• Ideal traits: highly polymorphic, codominant, reproducible, abundant, genome-wide, and neutral to environment.
• Trade-off: no single system meets all; choice depends on cost, throughput and prior sequence knowledge.

B. RFLP — Restriction Fragment Length Polymorphism

Polymorphism arises from differences in restriction-site position that alter fragment lengths.

• Method: digest genomic DNA with a restriction enzyme (e.g. EcoRI), separate fragments by electrophoresis, Southern-blot to membrane, hybridise with a labelled probe.
• Basis of variation: mutation creating or abolishing a recognition site, or insertions/deletions between sites.
• Inheritance: codominant—both alleles detected.
• Drawbacks: needs large amounts of pure DNA, is laborious, uses radioactive/probe labelling; no PCR required.

C. RAPD — Random Amplified Polymorphic DNA

Short arbitrary primers amplify anonymous genomic regions to give a multi-band fingerprint.

• Primer: single ~10-mer of arbitrary sequence, no target knowledge needed.
• Basis of variation: presence/absence of primer-binding sites; a band appears only when two sites lie in inverted orientation within amplifiable distance.
• Inheritance: dominant—band present or absent.
• Limitation: low reproducibility, sensitive to reaction conditions (Mg²⁺, template purity, cycler model).

D. AFLP — Amplified Fragment Length Polymorphism

Selective amplification of restriction fragments combines RFLP's reliability with PCR's power.

• Steps:
  • Digestion: two enzymes, a rare cutter (EcoRI) and a frequent cutter (MseI).
  • Adaptor ligation: known double-stranded adaptors ligated to fragment ends.
  • Selective amplification: primers complementary to adaptor + 1–3 selective nucleotides amplify a subset of fragments.
• Readout: high-resolution polyacrylamide gel, 50–100 bands per reaction.
• Inheritance: predominantly dominant; highly reproducible and high-throughput.

E. SSR — Simple Sequence Repeats (microsatellites)

Tandem repeats of 1–6 bp vary in copy number between genotypes.

• Example motif: (GA)n or (CAG)n; alleles differ by number of repeat units, e.g. (GA)₁₂ versus (GA)₁₅.
• Method: locus-specific primers flanking the repeat amplify a fragment whose length reflects repeat number.
• Inheritance: codominant, highly polymorphic, locus-specific and reproducible.
• Requirement: prior sequence to design flanking primers.

F. ISSR — Inter-Simple Sequence Repeats

A single repeat-anchored primer amplifies the region between two nearby microsatellites.

• Primer: built on a repeat motif with a short anchor, e.g. (AG)₈YC, where Y is a degenerate base fixing the 3′ end.
• Basis of variation: distance and presence of repeat loci flanking an amplifiable segment.
• Inheritance: mostly dominant; needs no prior sequence yet is more reproducible than RAPD.

G. SNP — Single Nucleotide Polymorphism

Variation at a single base is the most abundant marker in any genome.

• Nature: a one-base substitution, e.g. an A/G difference at a defined position.
• Detection: allele-specific PCR, sequencing, or high-throughput arrays.
• Inheritance: codominant, biallelic, amenable to automation and very high density.

IV. Molecular analysis of given plant DNA using DNA-based markers

A. Workflow from tissue to profile

The practical exercise moves a plant sample through a fixed sequence of steps.

• Sampling: fresh young leaf collected and, if needed, stored at −80 °C or in silica.
• DNA isolation: CTAB method—grind in cetyltrimethylammonium bromide buffer, chloroform:isoamyl alcohol extraction, isopropanol precipitation, 70 % ethanol wash.
• Quality/quantity check:
  • Gel: intact high-molecular-weight band on 0.8 % agarose confirms non-degraded DNA.
  • Spectrophotometry: A₂₆₀/A₂₈₀ ratio of 1.8–2.0 indicates protein-free DNA; A₂₆₀/A₂₃₀ ≈ 2.0 indicates no polysaccharide/phenol carryover.
• Amplification: set up PCR with the chosen marker's primers (RAPD, ISSR, SSR, etc.) and cycle as in Section II.
• Electrophoresis: run products—agarose (1.5–2 %) for RAPD/ISSR/SSR, polyacrylamide for AFLP/fine SSR—stain with ethidium bromide, visualise under UV against a size ladder.

B. Data scoring and analysis

Band patterns are converted into a numeric matrix for genetic interpretation.

• Binary scoring (dominant markers): each band scored 1 = present, 0 = absent per genotype, giving a 0/1 matrix.
• Allele sizing (codominant markers): SSR/SNP alleles recorded by fragment size or base identity, so heterozygotes show two peaks/bands.
• Similarity coefficient: genetic similarity computed, e.g. Jaccard's:

J = a / (a + b + c)

where a = bands shared by both samples, b = bands only in sample 1, c = bands only in sample 2.

• Clustering: a distance matrix (1 − J) feeds UPGMA to build a dendrogram grouping genotypes by relatedness.
• Polymorphism information content (PIC): measures a marker's discriminating power; higher PIC means the marker distinguishes more genotypes.

C. Applications and limitations

Marker analysis of plant DNA underpins several breeding and diversity tasks.

1. Applications:
   • Genetic diversity/fingerprinting: distinguishing cultivars and detecting adulteration.
   • Variety identification and purity testing: confirming hybrid seed identity.
   • Marker-assisted selection: tracking a trait linked to a codominant marker (SSR/SNP) across generations.
   • Phylogenetic and QTL mapping: placing loci on linkage maps.
2. Limitations:
   • Dominant systems (RAPD, ISSR, AFLP): cannot resolve heterozygotes; RAPD reproducibility is low.
   • Codominant systems (SSR, SNP): demand prior sequence and higher setup cost.
   • Common pitfall: poor-quality template (polyphenol contamination) causes failed or artefactual amplification, so the A₂₆₀/A₂₈₀ check is decisive before any marker assay.