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

•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.

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

Chloroplast

• organelle found in plant cells and eukaryotic algae

• conduct Photosynthesis• Chloroplasts are green....chlorophyll

pigment

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

PROPERTIES of ctDNA:i. Non- mendelian inheritanceii. Self replicationiii. Somatic segregation in plantsiv. Inherited independently of nuclear

genesv. Conservative rate of nucleotide substitution

enables to resolve plant phylogenetic relationships at deep levels of evolution.eg. familial level; mono- dicotyledonous

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)

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.

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.

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).

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 promising gene in this regard.

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.

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.

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

Analysis of noncoding regions of cpDNA could extendthe utility of the molecule at lower taxonomic levels.These zones tend to evolve more rapidly than do codingsequences, by the accumulation of insertions/deletionsat a rate at least equal to that for nucleotide substitutions(Curtis and Clegg 1984; Wolfe et al. 1987; Zurawski andClegg 1987; Clegg and Zurawski 199 1 ), and thereforethey can become very useful below the family level.

Noncoding regions of the chloroplast genome tend to evolve more rapidly than do coding regions

• 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

•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

Opportunities in plant molecular evolutionBroadly 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.

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

THANK YOU

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 SequencesLudovic Gielly and Pierre Taberlet

Molecular Systematics of Plants Pamela S. Soltis, Douglas E. Soltis, and Jeff]. Doyle

References