DNA barcode is a unique pattern or DNA sequence that identifies each living things. Barcoding relies on short, highly variably regions of the genome. With thousands of copies per cell, mitochondrial and chloroplast sequences are readily amplified by PCR, even from very small or degraded species. The chloroplast genome (cpDNA) of plants has been a focus of research in plant molecular evolution and systematics. The genome is small and constitutes an abundant component of cellular DNA. The chloroplast genome has been extensively characterized at the molecular level providing the basic information to support comparative evolutionary research. The rates of nucleotide substitution are relatively slow and therefore provide the appropriate window of resolution to study plant phylogeny at deep levels of evolution. A region of chloroplast gene rbcL–RuBisCo large subunit–is used for barcoding plants. The most abundant protein on Earth, RuBisCo (Ribulose-1,5-biphophate carboxylase oxygenase) catalyzes the first step of carbon fixation via Calvin cycle. It is the most prevalent conformation, found in most proteobacteria, cyanobacteria, algae and higher plants. As evolutionary rate of rbcL is suitable for study of phylogeny, it is often used as model for phylogenetic investigation. The most common gene used to provide sequence data for plant phylogenetic analyses is the plastid-encoded rbcL gene. The rbcL gene is located on cp genome as a single copy gene and has an enormous phylogenetic utility.
Understanding of these evolution patterns mat shed light on functional or structural features governing RuBisCo activity. The single copy rbcL gene is approximately 1430 base pairs in length, is free from length mutations except at the far 3′ end, and has fairly conservative rate of evolution. The sequence data of the rbcL gene are widely used in the reconstruction of phylogenies throughout the seed plants. The rbcL gene was one of the first plant genes to be sequenced and is still among the most frequently sequenced segments of plant DNA. Determination of phylogenetic relationship of various organisms is a difficult job as the living world exhibits unimaginable diversity with respect to its species content. About 500 rbcL sequences were used to address phylogenetic relationships within angiosperms and secondarily among extant seed plants. However, it is apparent that the ability of rbcL to resolve phylogenetic relationships below the family level is often poor. Patterns of amino acid replacement in the rbcL gene also reveal substantial variation in site-dependent probabilities of substitution. These complexities in mutational change should motivate the development of more realistic algorithms for phylogenetic inference when based on molecular data. Bousquet et al. (1992) found extensive variation in the evolutionary are of rbcL chloroplast gene sequences among seed plants, which they suggested may arise through differences in speciation rates. Chase et al. (1993) combined a largescale rbcL gene phylogeny of seed plants with the development of methods to analyse patterns of diversification within phylogenies.
Barraclough et al. (1996) found that there was a positive correlation between rate of rbcL sequences evolution and species diversification in angiosperms. This finding reflected a general relation between the rate of chloroplast DNA evolution and species diversification in flowering plants. Doyle et al. (1997) performed the phylogenetic analyses of the chloroplast-encoded rbcL gene in Leguminosae to find the evolution of nodulation. The comprehensive phylogenetic study of legume rbcL sequences results into the subfamilial and tribal taxonomy of the family. rbcL sequences prove as a source of phylogenetic characters in legumonosae which has proven useful as a means of constructing phylogenetic hypothesis. The rbcL results include a basal trichotomy among the groups of very unequal size. For example, Polhill et al. (1981) suggests an evolutionary trichotomy among Cercideae, elements of Cassieae, and the remainder of the family. Cercideae is a distinctive tribe with unique leaf and seed features and a putatively primitive base chromosome number (x=7), which could represent plesiomorphic features. The topology of the rbcL tree contains features that are in substantial agreement with many current hypotheses of legume relationships. The rbcL genes expression ability as predicted by CAI (codon adaptation index) is similar for most of the species.
A phylogenetic reconstruction based on a plastid gene not only provides an alternative for evolutionary studies of the organisms, but it also allows comparisons of phylogenies from host cells and their plastids. Furthermore, phylogenetic hypotheses based on chloroplast-encoded genes make it possible to study evolutionary origin of algal plastids too.
The chloroplast genome has provided such data, but the weak support for many clades in the rbcL tree suggests that data from additional genes will be required to construct rigorous hypotheses for some groups. Additional problems with constructing phylogenetic trees from rbcL sequences may be caused by RNA editing, pseudogenes, unequal rates of evolution, and inadequate taxon sampling. Data from phylogenetically independent nuclear genes will be of particular interest.
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