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 present in a study group (population, species, group of species).
  • Genetic diversity is more than just phenotypic diversity.

Traditional Molecular Markers

  • Based on Polymerase Chain Reaction (PCR) - amplification of ‘target’ DNA.
  • Mitochondrial DNA (mtDNA) – animals.
  • Chloroplast DNA (cpDNA) - plants.
  • Nuclear DNA (nDNA) – chromosomes.
  • Used in barcoding (species identification).

Polymerase Chain Reaction (PCR)

  • Step 1: DNA is heated to 95∘C95^\circ C to separate the double DNA strand.
  • Step 2: DNA is cooled to approximately 55^\\circ C for primers to bind to the DNA template. Oligonucleotide primers are used.
  • Step 3: Polymerase enzyme adds bases complementary to the DNA template, creating 2 identical copies of the original template.
  • Step 4: With each cycle, the number of copies of the DNA fragment doubles.
  • After 30 cycles, there are > 1 million copies of the DNA fragment.
  • The process is an exponential function of the type 2N2^N, where NN is the 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
  • Approximately 16Kbp in animals: 2 rRNA, 22 tRNA, 13 protein, Control Region

Advantages and Disadvantages

  • Advantages:
    • Easy to isolate and amplify.
    • Good for evolutionary studies.
    • Different genes evolve at different rates for different taxonomic levels (order-individual).
    • Useful for phylogeny and phylogeography.
  • Disadvantages:
    • Only informative for maternal dispersal and evolution.
    • Copies also found in the chromosomes.
    • Heteroplasmy or different mitochondrial sequences in different tissues is rare but problematic when it occurs.

Nuclear DNA

  • 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).
  • Whole genomes can be analyzed.

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 and Disadvantages of AFLP

  • 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: need high quality DNA.
    • Now replaced by PCR-based fingerprinting (AFLP) – traditional fingerprinting was based on hybridization of probes

Sanger Sequencing (termination method)

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

Shotgun Sequencing

  • Involves fragmentation, shotgun rounds, and stitching the consensus sequence together.
  • Pair(wise) End Sequencing allows for sequencing of very long sequences (thousands of bp long).

2nd Generation Sequencing

  • The technology improved in terms of miniaturization.
    • Reactions are now done in picoliters (10−1210^{-12} liters) instead of 0.2 ml volumes (microcentrifuge tube).
  • Camera Imaging.

Illumina

  • Fragmentation of DNA for sequencing (e.g., sonication).
  • Tagging = attachment (ligation) of adaptor to DNA fragments
  • Attachment of adaptor ligated DNA fragments to surface of flow cell
  • “Bridge” amplification and denaturation cycles (similar to PCR), producing “clusters”
  • DNA sequencing of clusters with fluorescently labeled dNTPs
  • Company: Illumina®
  • Fragment length: 2*150 bases (pair-end).
  • Number of reads per run: 2(310910^9) – 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
    • Pacific BioSciences - PacBio
    • NANOPORE
    • Oxford NanoPore

Summary

  • Technological developments during the past 60 years have enabled studying DNA.
  • Mitochondrial DNA enables studying maternal histories, while nuclear DNA provides information about both the maternal and paternal lineage.
  • Mitochondrial DNA is effectively 1 locus, while the nuclear DNA has millions of independent loci.
  • Recent technological developments (i.e., miniaturization and high-resolution imaging) in the past 20 years have allowed a revolution in sequencing technologies.
  • Technologies are relatively short-lived (even if developing costs millions of £/€/$).
  • Illumina has almost taken over all the market with Genome Analyzer (GAI and II) and HiSeq machines.
  • Sequencing technologies use a variety of approaches to identify different base pairs, e.g., fluorochromes, ionic charge changes, membrane conductivity.