Chloroplast DNA and the study of plant phylogeny- present status and future prospects

1. Chloroplast DNA and the study
of plant phylogeny- present
status and future prospects

3. •The increasing available completely sequenced organisms and the importance of
evolutionary processes that affect the species history, have stressed the interest in
studying the molecular evolution events at the sequence level.
Molecular evolution
•The field of molecular evolution developed rapidly into a significant area of
research activity in the late 1960s
•Owning to the development of the recombinant DNA technology and the invention
of rapid DNA sequencing methods the 1980s has seen the explosion in the use of
molecular data for the study of evolutionary problems
•The field of plant molecular evolution has participated in this expansion, especially
where the chloroplast genome is concerned.

4. Applications:
Molecular evolution analysis has clarified:
• the evolutionary relationships between humans and other
primates;
• the origins of AIDS;
• the origin of modern humans and population migration;
• speciation events;
• genetic material exchange between species.
• origin of some deseases (cancer, etc...)
Molecular evolution

5. Chloroplast
• organelle found in plant cells and
eukaryotic algae
• conduct Photosynthesis
• Chloroplasts are
green....chlorophyll pigment

6. Chloroplast genome
 Chloroplast DNA (cpDNA) is also known as
plastid DNA (ptDNA).
 Circular double stranded DNA molecule
 Ct genomes are relatively larger
 140kb in higher plants.
 200kb in lower eukaryotes.
 Multiple copies of genome per organelle.
Vary in size ,
 But are large enough to code 50-100
proteins as well as rRNAs & tRNAs

7. PROPERTIES of ctDNA:
i. Non- mendelian inheritance
ii. Self replication
iii. Somatic segregation in plants
iv. Inherited independently of nuclear
genes
v. Conservative rate of nucleotide substitution
enables to resolve plant phylogenetic
relationships at deep levels of evolution.
eg. familial level; mono- dicotyledonous

8. cpDNA regions includes Large Single-Copy
(LSC) & Small Single-Copy (SSC) regions,
and Inverted Repeats (IRA & IRB).
Variation in length mainly due to
presence of inverted repeat (IR)

9. GENOME SEQUENCING:
• Two of the first ct genome sequenced
- liverwort, Marchantia polymorpha.
- tobacco, Nicotiana tabacum.
• Tobacco DNA is larger , contains 150 genes
and that of liverwort is 134.

10. The chloroplast genome is well studied for evolutionary and phylogenetic study,
because cpDNA is a relatively abundant component of plant total DNA thus
facilitating the extraction and analysis
They have an extensive background of molecular information on the chloroplast
genome. The complete DNA sequences of three cpDNA are known (the liverwort
Marchantia polymorpha, tobacco – Nicotina tobacum, and Rice – Oryza sativa
Another advantage of the chloroplast genome for the evolutionary research is a
conservative rate of nucleotide substitution with a technical and a fundamental
advantage
Why chloroplast genome ?
Because of its complexity and repetitive properties, the nuclear genome is used in
systematic botany less frequently.

11. Molecular Systematics on cpDNA
 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.
cpDNA is an extremely valuable molecule for studying phylogenetic
relationships between closely related species (Palmer 1987; Palmer et
al. 1988; Clegg et al. 199 1).

12. The most common gene used to provide sequence data for plant phylogenetic
analyses is the plastid-encoded rbcL gene (Chase et al., 1993; Donoghue et al., 1993).
This single copy gene is approximately 1430 base pairs in length, is free from length
mutations except at the far 3' end, and has a fairly conservative rate of evolution.
The function of the rbcL gene is to code for the large subunit of ribulose 1, 5
bisphosphate carboxylase/oxygenase (RUBISCO or RuBPCase).
The sequence data of the rbcL gene are widely used in the reconstruction of
phylogenies throughout the seed plants. However, it is apparent that the ability of
rbcL to resolve phylogenetic relationships below the family level is often poor
(Doebley et al., 1990).
Thus, interest exists in finding other useful DNA regions that evolve faster than does
rbcL to facilitate lower-level phylogenetic reconstruction. The matK gene is a

13. Direct sequencing of polymerase chain reaction products is
now an expanding area of plant systematics and evolution.
Within angiosperms the rbcL gene has been widely
sequenced and used for inferring plant phylogenies at higher
taxonomic levels. Unfortunately rbcL does not usually
contain enough information to resolve relationships
between closely related genera, such as Hordeum, Triticum,
and Aegilops. One solution to this problem could be to
analyze noncoding regions of chloroplast DNA, which are
supposed to evolve more rapidly than coding regions.

14. Four main approaches employ the chloroplast genome to infer
relationships:
(1) restriction site analysis;
(2) structural changes in the chloroplast genome, including inversions,
large deletions, and the loss of specific introns and genes;
(3) comparative DNA sequencing; and
(4) PCR based approaches.

15. Chloroplast Gene Evolution
• In most respects, the molecular evolution of chloroplast
genes mirrors that of nuclear genes
• Chloroplast protein encoding genes evolve at a rate that is
on average fivefold lower than plant nuclear genes
• Reduced rate of cpDNA gene evolution arises from a
reduced mutation rate

16. Analysis of noncoding regions of cpDNA could extend
the utility of the molecule at lower taxonomic levels.
These zones tend to evolve more rapidly than do coding
sequences, by the accumulation of insertions/deletions
at a rate at least equal to that for nucleotide substitutions
(Curtis and Clegg 1984; Wolfe et al. 1987; Zurawski and
Clegg 1987; Clegg and Zurawski 199 1 ), and therefore
they can become very useful below the family level.
Noncoding regions of the chloroplast genome tend to
evolve more rapidly than do coding regions

17. • Sequencing studies of cpDNA genes have utilized both the chemical
cleavage reactions applied to the end labeled DNA fragments and
dideoxy chain-termination reactions applied to DNA clones in double
stranded or single stranded vectors
•In comparative sequencing studies, the conservative nature of cpDNA
evolution facilitated the development of sets of synthetic DNA
primers, which is now used with the dideoxy method to sequence
specific genes rapidly from diverse plant species
DATA ACQUISITION

18. •Recently these primers have been used with PCR technology to
further simplify comparative cpDNA analysis
•The development of the technologies like PCR , DNA sequencing and
genomic sequencing had made a useful method for comparitative
studies

19. Opportunities in plant molecular evolution
Broadly speaking, research opportunities exist in the
following three areas:
(1) studies of the mechanisms of gene and genome
evolution,
(2) the investigation of plant phylogeny, and
(3) the use of fossil DNAs to study rates of evolution
and to characterize paleocommunities.

20. Problems in plant molecular evolution
• The major problem concerns the management and analysis of DNA sequence data
• A potential future problem is the sharing of sequence information with other
members of the interested research community
• There are at least two journals [ Nucleic Acids Research and Plant Molecular
Biology) with incorporated sections where one can report nucleic acid sequence
data with little or no comment. This is valuable for the evolutionary community
Because it provides a mechanism for the sharing of data
• A second issue which has been the subject of much discussion is the choice of
appropriate methods of data analysis

21. THANK YOU

22. Chloroplast DNA and Molecular Phylogeny Jeffrey D. Palmer
Chloroplast DNA Variation and Plant Phylogeny Author(s): Jeffrey D. Palmer,
Robert K. Jansen, Helen J. Michaels, Mark W. Chase andJames R. Manhart
The Use of Chloroplast DNA to Resolve Plant Phylogenies: Noncoding versus rbcL Sequences
Ludovic Gielly and Pierre Taberlet
Molecular Systematics of Plants Pamela S. Soltis, Douglas E. Soltis, and Jeff]. Doyle
References