Chloroplast DNA in Molecular Systematics

-organelle found in plant cells and eukaryotic algae -Photosynthesis Chloroplast

- Chloroplast DNA (cpDNA) is also known as plastid DNA (ptDNA). - Circular double stranded DNA molecule - Chloroplast genome size ranges kb with majority of plants fall into kb. (Pelargonium has a chloroplast genome size 217kb) - contain about 100 genes to synthesize proteins - cpDNA regions includes Large Single-Copy (LSC) & Small Single-Copy (SSC) regions, and Inverted Repeats (IR A & IR B ). - Conifers and a group of legumes lack Inverted Repeats. Chloroplast genome

IR A IR B LSC SSC

- Complete chloroplast DNA sequences of four land plants (Nicotiana tabacum, Marchantia polymorpha, Oryza sativa and Epifagus virginiana) were available for comparative study on structure and gene content of chloroplast genomes in 1980s. - At present, the number of complete chloroplast genome sequences is 122 (from 114 different organisms). eg. Arabidopsis thaliana, Coffea arabica, Eucalyptus globulus, Glycine max, Gossypium hirsutum, Helianthus annuus, Lycopersicon esculentum, Nymphaea alba, Phaseolus vulgaris, Pinus koraiensis, Piper cenocladum, Solanum tuberosum, Triticum aestivum, Vitis vinifera, Zea mays etc. Chloroplast genome

- cpDNA is a relatively abundant component of plant total DNA, thus facilitating extraction and analysis. - Conservative rate of nucleotide substitution enables to resolve plant phylogenetic relationships at deep levels of evolution. eg. familial level; mono- & dicotyledonous; - Chloroplast protein-coding genes evolve at a rate that is on average fivefold slower than plant nuclear genes. Characteristics of Chloroplast Genome

- Strictly maternally inherited in most angiosperms while in conifers, inheritance is paternal. - Chloroplast DNA is passed on from one generation to the next with only an occasional mutation altering the molecule; sexual recombination does not occur. Characteristics of Chloroplast Genome

- cpDNA regions can be amplified by means of PCR. - The resulted PCR products may be subjected to RFLP or DNA sequencing. - Common cpDNA regions used in systematic study: rbcL (1400bp), trnL-trnF ( bp), atpB- rbcL (1000bp), trnL intron (300bp), matK (2600bp), trnT-trnL ( bp), 16S (1400bp), rpoC (3600bp) etc. Molecular Systematics on cpDNA

IR A IR B LSC SSC rbcL matK atpB-rbcL trnL intron trnL-trnF trnT-trnL rpoC 16S

- Restriction site mapping of the entire chloroplast genome. (involve the isolation of chloroplast DNA from the total DNA) Molecular Systematics on cpDNA The whole chloroplast genomes of different Brassica species were digested with SacI

- Singular structural rearrangements (e.g. inversions and intron losses). - Loss of intron of rpl2 gene was found in species of order Caryophyllales (cacti, amaranths, carnations, carnivorous plants). Molecular Systematics on cpDNA

- On of the most comprehensive phylogenetic study of cpDNA rearrangement involved a 22kb inversion found to be shared by 57 genera representing all tribes of the family Asteraceae (sunflowers), a large plant family with 20,000 species and 1100 genera. - 50kb inversion brought psbA closer to rbcL in legumes. - 25kb inversion brought atpA closer to rbcL in wheat. Molecular Systematics on cpDNA

H. nervosa H. dyeri H. dryobalanoides H. beccariana H. pierrei H. latifolia H. mengerawan H. myrtifolia H. ferruginea H. sangal H. nutans H. odorata H. helferi H. apiculata H. wightiana Neobalanocarpus heimii Subsection Hopea Subsection Dryobalanoides Subsection Sphaerocarpae Subsection Pierrea Hopea clade Dryobalanoides clade Outgroup 72-bp deletion in the trnL-trnF HopeaHopea DryobalanoidesDryobalanoides Phylogeny based on the trnL-trnF, trnT-trnL and atpB-rbcL sequences. Upuna borneensis H. subalata H. bilitonensis Tree length = 143 CI = RI = H. pubescens