Chloroplast DNA in Molecular Chloroplast DNA in Molecular SystematicsSystematics

- organelle found in plant cells and eukaryotic algae

- Photosynthesis

ChloroplastChloroplast

- Chloroplast DNA (cpDNA) is also known as plastid DNA (ptDNA).

- Circular double stranded DNA molecule

- Chloroplast genome size ranges 120-217kb with majority of plants fall into 120-160kb. (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 (IRA & IRB).

- Conifers and a group of legumes lack Inverted Repeats.

Chloroplast genomeChloroplast genome

IRA

IRB

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 genomeChloroplast 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 GenomeCharacteristics 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 GenomeCharacteristics 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 (250-800bp), atpB-rbcL (1000bp), trnL intron (300bp), matK (2600bp), trnT-trnL (400-800bp), 16S (1400bp), rpoC (3600bp) etc.

Molecular Systematics on cpDNAMolecular Systematics on cpDNA

IRA

IRB

LSC

SSC

rbcL

matK

atpB-rbcL

trnL intron

trnL-trnF

trnT-trnLrpoC

16S

- Restriction site mapping of the entire chloroplast genome. (involve the isolation of chloroplast DNA from the total DNA)

Molecular Systematics on cpDNAMolecular 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 cpDNAMolecular 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 cpDNAMolecular Systematics on cpDNA

H. nervosaH. 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

69

84

89

100

75

5192

Subsection Hopea

Subsection Dryobalanoides

Subsection Sphaerocarpae

SubsectionPierreaHopea clade

Dryobalanoides clade

Outgroup

72-bp deletionin the trnL-trnF

Hopea

Dryobalanoides

Phylogeny based on the trnL-trnF, trnT-trnL and atpB-rbcL sequences.

Upuna borneensis

H. subalata

72

96

H. bilitonensis

Tree length = 143CI = 0.8811RI = 0.8651

H. pubescens