Molecular Markers and Genetic Variation

Phenotype to Genotype

  • Molecular markers are used to study genetic variation.

  • Genetic diversity is the variety of alleles and genotypes within a study group (population, species, or group of species).

  • Genetic diversity is more than just phenotypic diversity.

Traditional Molecular Markers

  • Based on Polymerase Chain Reaction (PCR) for amplifying target DNA.

    • Mitochondrial DNA (mtDNA) in animals.

    • Chloroplast DNA (cpDNA) in plants.

    • Nuclear DNA (nDNA) in chromosomes.

  • Used in barcoding (species identification).

Polymerase Chain Reaction (PCR)

  • Step 1: DNA is heated to 95°C to separate the double DNA strand.

  • Step 2: DNA is cooled to approximately 55°C, allowing primers to bind to the DNA template (Oligonucleotide primers).

  • Step 3: Polymerase enzyme adds bases complementary to the DNA template, creating two identical copies of the original template.

  • Step 4: With each cycle, the number of DNA fragment copies doubles.

  • After 30 cycles, there are >1 million copies of the DNA fragment.

  • This is an exponential function of the type 2N2^N where NN = number of PCR cycles.

Mitochondrial DNA (mtDNA)

  • Very numerous inside each cell.

  • Has its own circular DNA.

  • Multiple copies (>300 per cell).

  • Haploid (one copy), clonal inheritance – maternal.

  • Non-recombinant.

  • Evolves ~10x faster than the nuclear genome.

  • Contains genes such as CytB, COX3, COX2, COX1, NADH2, NADH1, NADH5, 16S RNA, D-Loop, 12S RNA, NADH6, NADH4, ATP6, ATP8, NADH4L, NADH3, rRNA, tRNA, ATP Synthase, Cytochrome Oxidase, Cytochrome bc1, and NADH: ubiquinone Oxidoreductase.

  • ~16Kbp in animals: 2 rRNA, 22 tRNA, 13 protein, Control Region.

Advantages:

  • Easy to isolate and amplify.

  • Good for evolutionary studies because different genes evolve at different rates suitable for different taxonomic levels (order-individual).

  • Useful for phylogeny and phylogeography.

Disadvantages:

  • Only informative for maternal dispersal and evolution.

  • Copies can also be found in the chromosomes.

  • Heteroplasmy (different mitochondrial sequences in different tissues) is rare but problematic when it occurs.

Nuclear DNA (nDNA)

  • Huge genome – Billions of base pairs.

  • Chromosomal.

  • Bi-parental inheritance.

  • Provides independent information for trees, population genetics, and parentage.

  • Includes Single Nucleotide Polymorphisms (SNPs) and Copy Number Variants (CNVs).

  • Allows whole genome sequencing.

Methods Available:

  • Classical DNA fingerprinting.

  • PCR-based fingerprinting.

  • AFLP – amplified fragment length polymorphism.

  • RAPD – random amplified polymorphic DNA.

  • DNA profiling (using known genomic positions).

  • Microsatellites.

  • SNP – single nucleotide polymorphism.

  • DNA sequencing.

  • High throughput, massively parallel sequencing.

Advantages:

  • Highly variable bands in a bar-code.

  • Lots of genomic locations (loci) assessed at the same time.

  • Gives phylogenetics and population analysis much more statistical power.

  • No prior knowledge of genome required.

Disadvantages:

  • Cannot identify individual loci or type of inheritance (biparental, maternal, alleles).

  • Unable to use non-invasive sampling; requires high-quality DNA.

  • Traditional fingerprinting, based on hybridisation of probes, has been replaced by PCR-based fingerprinting (AFLP).

Sanger Sequencing (Termination Method)

  • Limited to relatively short sequences (~800bp).

Shotgun Sequencing

  • Involves fragmenting the DNA into multiple small pieces

  • Sequencing each fragment separately

  • Stitching the consensus sequence back together for the full sequence

  • Pair(wise) End Sequencing enables the sequencing of very long sequences (thousands of bp long)

2nd Generation Sequencing

  • Revolutionized sequencing, with multiple revolutions occurring in a single decade.

Reasons for Revolution:

  • Miniaturization: Reactions performed in picoliters (10-12 liters).

  • Camera Imaging: Allows for high-throughput analysis, such as with 454 Life Technologies and Illumina.

Illumina

  • Fragmentation of DNA via sonication.

  • Tagging: attachment (ligation) of adaptor to DNA fragments.

  • Attachment of adaptor ligated DNA fragments to the surface of flow cell.

  • “Bridge” amplification and denaturation cycles (similar to PCR), producing “clusters”.

  • DNA sequencing of clusters with fluorescently labelled dNTPs.

  • Company: Illumina®

  • Fragment length: 2*150 bases (pair-end)

  • Number of reads per run: 2(3109) – two flow cells

  • Total output per run: 1.6-1.8 Tb

  • Time per run: < 3 days

3rd Generation Sequencing

  • Single Molecule Real Time (SMRT) sequencing : light - conductivity.

  • Pacific BioSciences (PacBio).

  • Oxford NanoPore Technologies.