1. Molecular Techniques in Molecular Systematics

2. DNA-DNA hybridisation -Measures the degree of genetic similarity between pools of DNA sequences. -Normally used to determine the genetic distance between two species. -The method compares the melting of a labeled sample after it is hybridized to iteself vs its melting after hybridized to unlabeled DNA of another organism.

3. RAPD, AFLP, Minisatellites -Data is scored as presence (1) or absence (0) of a DNA band/fragment. -Suitable for studies involving closely related taxa because the variation detected by these markers is high. -Homology problem may arise due to co- migration of DNA bands/fragments. RADP data

4. PCR-RFLP / CAP -Data scored as presence (1) or absence (0) of a restriction site. -4-base and 6-base cutters are used to generate data. -Data scoring becomes difficult if the variations involve length mutations (deletions or insertions / indels)

5. CAPS 580 bp 290 bp 180 bp 580 bp 180 bp 290 bp PCR products of the trnS-trnM region (cpDNA) restricted by TaqI

6. PCR-RFLP / CAP -The probability of restriction site loss is higher than restriction site gain. -A site loss could be due to various character- states (conditions). Taxon Species A Species B Species C Species D Species E Sequence CGTATTTAAACCGCTC CGTATTTAAACCGCTC CGTATTCAAACCGCTC CGTATTGAAACCGCTC CGTATTTCAACCGCTC Restriction Cut Cut No Cut No Cut No Cut Restricted by DraI (TTTAAA)

7. DNA Sequencing -DNA data is multiple state data. It normally exist in 4 different bases (A, T, C and G). -DNA data must be aligned (multiple sequence alignment) in order to be scored. -CLUSTAL software is used to align DNA sequences.

8. DNA Sequencing Multiple Sequence Alignment using CLUSTAL

9. DNA Sequencing -The evolution of DNA sequences is various depending on DNA regions (protein coding region, non-protein coding region, intergenic spacer, intron, repetitive DNA region etc.) -For protein coding region, the mutation rate for synonymous base substitution is higher than non-synonymous base substitution. Taxon Species A Species B Species C DNA Sequence TTT GTT TGG TTT GTA TGG TTT TTT TCG Amino acid sequece Phe-Val-Trp Phe-Val-Trp Phe-Phe-Trp

10. Amino Acid Sequencing -Amino acid sequencing was practiced before the establishment of DNA sequencing methods. -Amino acid data is a multiple state data. It consists of 20 states (20 different amino acids).

11. Single-Letter Amino Acid Code G - Glycine (Gly) P - Proline (Pro) A - Alanine (Ala) V - Valine (Val) L - Leucine (Leu) I - Isoleucine (Ile) M - Methionine (Met) C - Cysteine (Cys) F - Phenylalanine (Phe) Y - Tyrosine (Tyr) W - Tryptophan (Trp) H - Histidine (His) K - Lysine (Lys) R - Arginine (Arg) Q - Glutamine (Gln) N - Asparagine (Asn) E - Glutamic Acid (Glu) D - Aspartic Acid (Asp) S - Serine (Ser) T - Threonine (Thr)

12. Amino Acid Sequencing -Amino acid sequencing is time consuming. It is done by HPLC approach. -At present the amino acid sequences are generated mainly from the inference of protein coding DNA sequences. -Amino acid of hemoglobin (in animals) and amino acid of large subunit ribulose-1,5-bisphophate carboxylase (rbcL; in plants) are widely used for phylogenetic studies.