0976-7908 Yadav et al www.pharmasm.com IC

Vol - 4, Issue - 4, Jul-Sept 2013

ISSN: 0976-7908

Yadav et al

PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES

MOLECULAR MARKERS OF AROMATIC AND MEDICINAL PLANTS: A REVIEW Rajesh Kumar1, Shiv S Yadav1*, Shiv K Mishra2, Anjani K Srivastav3, Madhu Tripathi1 1

Aquatic Toxicology Research Laboratory Department of Zoology, University of Lucknow, Lucknow School of Biotechnology, RGPV, Bhopal (M.P.) 3 Institute of Engineering and Technology (IET) Lucknow 2

ABSTRACT Molecular markers have been used to help to elucidate some aspects of genetic diversity in aromatic and medicinal plants, the genetic relationships between different cultivars and comparisons of molecular marker analysis to the chemical composition of plants. In this review an overview of the most important techniques involving molecular markers is given. A literature survey on molecular markers is presented with some examples of aromatic and medicinal plants. However an understanding of; what controls flavor and aroma production in aromatic plants is not an easy task to accomplish. Many facts of plant secondary metabolite and volatiles production in aromatic plants are still unknown. The way from genomics to proteomics is not well identified yet some research with model plants has already been performed. To justify the question of the synthesis of secondary metabolites different biochemical and genetic approach approaches is given and summarized in this review. Keywords: Genomics; Expressed sequence tags (ESTs); Molecular marker and aromatic plants. INTRODUCTION Aroma are unique characters that are determined by a complex mixture of low-molecular weight volatile molecules belonging to three major groups phenylpropanoids (including benzenoids), fatty acid derivatives and terpenoids[1]. Due to several factors such as the invisibility of this character the limited human sense of smell and the highly variable nature of the trait no simple efficient and reliable methods to screen for genetic variation have been developed. Traditionally systematic and phylogenetic analysis of aromatic plants was based on macroscopic and microscopic morphological characters. Later on the importance of secondary metabolite composition was recognized and nowadays chemotaxonomy has a considerable impact on plant systematic. However the distribution of secondary metabolites also reflects adaptations and quite often allelochemicals of high structural specificity occur simultaneously in unrelated families of plants [2]. More recently chemotaxonomy has been combined with molecular biology and the analysis of www.pharmasm.com

IC Value – 4.01

344


ISSN: 0976-7908

Yadav et al

either chloroplast or nuclear DNA sequences provide valuable contributions to the understanding of the phylogeny of a certain species. Flowery aroma is a highly variable trait. In many taxa, there are aromatic species that are closely-related to non-aromatic ones, leading to the conclusion that the ability to produce aroma is a trait that is easily acquired and easily lost [3]. Also, considering that flower aroma is a complex mixture of different compounds and that even two closely related species show distinctive mixtures of volatiles. It seems that aroma is an easily evolved trait. Advances in molecular genetics in the last 20 years have been facilitated by rapid DNA amplification techniques using polymerase chain reaction (PCR), by rapid sequencing methods (either manual or automated sequencing systems) and computer software programs. All these techniques have enabled the placement of molecular markers onto the maps of chromosomes of most major crop species and the subsequent tagging of genes of interest by their placement near those markers [4]. These important developments in crop species have been followed by some research in aromatic plants. Some important contributions can be found in the literature which reports the use of molecular markers to study aromatic plants and spices in families such as Apiaceae, Lamiaceae, Cupressaceae and Asteraceae. However basic research on fragrances is just beginning. For many years research into floral fragrance focused on its chemical elucidation coupled with chemical synthesis to produce the large quantities demanded by perfume industries. The search for elucidation of the pathways with respect to the enzymes and genes involved and the molecular mechanisms controlling them have only recently appeared. To date no convenient plant model systems that enable biochemical and forward and reverse genetics studies of aroma and flavor are available. In the case of aromatic plants a deeper insight into the genetics of the formation of plant secondary metabolites will lead to efficient breeding strategies assisted by molecular techniques. Also new tools such as genomics and proteomics are becoming more important tools in the study of secondary metabolism of aromatic plant. Genetic Markers Genetic markers are a valuable tool either in classical breeding or germplasm characterization and they can be defined as either morphological or molecular. The former has the advantage of being easily monitored without specialized biochemical or www.pharmasm.com

IC Value – 4.01

345


ISSN: 0976-7908

Yadav et al

molecular techniques. These markers have limited use because their number is very limited and also because they are altered by epistatic and pleiotropic interactions [5]. The advent of molecular markers overcame most of the problems associated with using morphological markers in which major phenotype-altering genes were used as genetic markers. Polymorphism is often much more easily and reliably detected at the molecular level than at the phenotypic level. These molecular markers can be either biochemical (Isozymes/allozymes) or DNA markers. Isozymes can be defined as multiple molecular forms of a single enzyme, usually having similar enzymatic properties but slightly different amino acid compositions as a result of differences in the nucleotide sequences that code for the proteins. These variant forms can be separated by electrophoresis due to the different electrophoresis mobility of the corresponding protein. Isozymes have the advantage of being co-dominant markers however they are limited in number and can also be modified due to post-translational processing [4,6]. There is often differential expression of isozymes in different tissues. As a result several samplings of the population may be necessary to score all the available isozymes. DNA Markers DNA markers offer several advantages such as being unlimited in number and unaffected by the environment. Also they are present in different tissues and at different ages of the individual. DNA markers are free of pleiotropic effects, allowing any number of markers to be monitored in a single population. DNA marker analysis can be carried out at any stage of the life cycle of an organism and from almost any tissue [5]. Most DNA markers are based on polymer chain reaction (PCR) amplification and require only a few nanograms of DNA for analysis. The process of PCR was first described in 1986 by Mullis and coworkers [7] and some years later in 1993 Mullis received the Nobel Prize in Medicine and Physiology for this discovery. PCR is a relatively simple process, in which DNA is amplified and virtually unlimited copies of selected DNA fragments can be generated in a short period of time. PCR provides an extremely sensitive means of amplifying small quantities of DNA and is routinely used nowadays to detect low levels of bacterial infection or rapid changes in transcription at the single-cell level, as well as the detection of specific individuals DNAs in forensic science. The development of PCR amplification resulted in an explosion of new techniques in molecular biology such as www.pharmasm.com

IC Value – 4.01

346


ISSN: 0976-7908

Yadav et al

RAPDs, AFLPs, ISSRs, ITSs, SSRs and SNPs and this is in fact an area where new concepts and terminology arise. Briefly there are different techniques which have been used to some extent in plant characterization. Restriction fragment-length polymorphisms (RFLPs) RFLPs are a result of polymorphisms in the length of the restriction fragments obtained by enzymatic digestion of the DNA [8]. The basic steps in RFLP analysis involve DNA extraction, restriction, digestion and Southern hybridization, using a probe that will anneal to the genome of interest. RFLP linkage maps have been constructed for many economically important crops, such as potato, maize, rice, lettuce, sunflower, sugar beet, barley and soybean, to give just a few examples but concerning aromatic plants and spices the literature is scarce or even lacking with regard to RFLPs. Random amplified polymorphic DNA (RAPD). RAPD involves PCR amplification of random DNA sequences from genomic DNA using short primers of arbitrary nucleotide sequence. This method was developed simultaneously in two independent laboratories and was named random amplified polymorphic DNA (RAPD) [9] and amplified polymorphic PCR [10]. Polymorphisms are a result of base pair substitutions or insertions/ deletions that modify the primer annealing site or insertions into the genomic sequence that separate the primer site to a distance that does not allow for amplification to occur. There are several advantages of RAPDs relative to other markers. They do not require previous knowledge of the genome as occurs with most other types of marker and it is not necessary to construct or maintain a genomic library as is the case for RFLPs. RAPDs provide an essentially unlimited number of markers throughout the genome and reveal high levels of polymorphism even within and among species that show little polymorphism using other markers. However problems related to lack of reproducibility have been reported by several authors [11–13]. Amplified fragment length polymorphisms (AFLPs). AFLP was first described by Vos and co-workers, who used different DNA sources such as yeast, Arabidopsis thaliana, Lycopersicum esculentum, Zea mays and human DNA to test the technique. Since then it has been extensively used in different plants and other organisms [14]. Polymorphisms arise due to single nucleotide change within restriction sites or adjacent nucleotides used for AFLP primer annealing. Polymorphisms can also result from deletions, insertions and rearrangements affecting the presence of restriction sites www.pharmasm.com

IC Value – 4.01

347


ISSN: 0976-7908

Yadav et al

and adjacent sequences. These markers are more informative than other DNA marker systems and typically generate 50–100 restriction fragments in each reaction resulting in a huge amount of loci scored per reaction.AFLP are quantitative markers and it is possible to differentiate the homozygous from the heterozygous genotype by comparing the intensity of the amplified bands. However AFLP markers can also be scored as dominant markers limiting the information they provide. Amplified fragment length polymorphisms (AFLPs) AFLP was first used in different DNA sources such as yeast, Arabidopsis thaliana, Lycopersicum esculentum, Zea mays and human DNA to test the technique. Since then it has been extensively used in different plants and other organisms [14]. Polymorphisms arise due to single nucleotide change within restriction sites or adjacent nucleotides used for AFLP primer annealing. Polymorphisms can also result from deletions, insertions and rearrangements affecting the presence of restriction sites and/or adjacent sequences. These markers are more informative than other DNA marker systems and typically generate 50–100 restriction fragments in each reaction resulting in a huge amount of loci scored per reaction. AFLP are quantitative markers and it is possible to differentiate the homozygous from the heterozygous genotype by comparing the intensity of the amplified bands. However AFLP markers can also be scored as dominant markers limiting the information they provide. There are several examples of the use of AFLP markers to study aromatic plants. Inter-sequence simple repeat (ISSR) ISSR technique is a PCR-based method which uses microsatellites as primers in a single reaction targeting multiple genomic loci mainly to amplify ISSR sequences of different sizes. The microsatellite repeats used as primers for ISSRs can be dinucleotide, trinucleotide, tetra nucleotide or pentanucleotide. The primers used can be either unanchored [15,16,17] or more usually anchored at 3’or 5’ end with 1 to 4 degenerate bases extended into the flanking sequences [18] .It is a quick and simple method that combines most of the advantages of microsatellites (SSRs) and amplified fragment length polymorphisms (AFLPs) to the universality of random amplified polymorphic DNA (RAPD). One major advantage of ISSRs is the high reproducibility they provide with www.pharmasm.com

IC Value – 4.01

348


ISSN: 0976-7908

Yadav et al

values over 99% being reported [19]. Decade ago, the internal transcribed spacers (ITS) of the nuclear ribosomal 18S–5.8S–26S cistron have been the most popular target region in the nuclear genome for evolutionary studies of diverse plant groups [20, 21]. Nucleotide substitutions or indels within the 5.8S region do not occur among sequences from the same individual [22, 23]. The 18S-5.8S-28S section of nuclear ribosomal DNA is known as the internal transcribed spacer region (ITS). ITS DNA sequences are widely used in plant molecular systematic to infer phylogenetic relationships [24, 25]. Simple sequence repeats (SSRs) SSRs or microsatellites are tandem repeats of simple sequences consisting most frequently of 2, 3 or 4 nucleotides (di- tri- and tetra nucleotides) that can be repeated 9-90 times [26]. The copy number of these repeats, which can be highly variable due to unequal crossing over, is the basis for the polymorphism. In plants, it has been demonstrated that microsatellites can be highly informative. The procedure for SSR marker development in crops, in the absence of substantial sequence data, includes the construction of a DNA library and screening of the library with probes corresponding to the repetitive sequences desired. In order to design unique primers flanking the sites of SSRs sequencing of the selected clones is required. Single-nucleotide polymorphisms (SNPs) SNPs arise from naturally occurring single-base changes or even single-base insertions or deletions although these are less frequent. SNPs hold great promise as markers in inbreeding crops or in experiments where the parents of a segregating cross are too closely related to permit efficient analysis using other molecular markers. One typical way in which SNPs have been identified is by comparing genomic sequence data to previously existing expressed sequence tag (EST) sequence (EST arises from cloned and sequenced cDNA obtained from mRNA extracted from various plant tissues. SNPs are an increasingly important molecular marker tool for the near future. However in research on aromatic plants and spices SNPs have not so far been used. Molecular Markers: Some Examples with Aromatic Plants and Spices In recent years, research in aromatic plants and spices has imported and exploited knowledge coming from other plant species, particularly concerning molecular markers. These studies have several goals. www.pharmasm.com

IC Value – 4.01

349


ISSN: 0976-7908

Yadav et al

(1) The assessment of genetic diversity in endangered species. (2) Genetic relationships between different cultivars (3) Comparison of the molecular marker analysis of the chemical composition of the plants. The most frequently used markers for these studies have been RAPD, AFLP ,ISSR and ITS, within such different families such as Apiaceae,Lamiaceae,Cupressaceae and Asteraceae.The family Apiaceae (Umbelliferae) provides a very good an example in which ITSs have been used thoroughly to help understanding of the systematic of this family on which numerous reports are available [27,28]. Concerning the combination of data coming from DNA fingerprinting using molecular markers and essential oil composition the results have varied from species to species and in the literature contradictory results can sometimes be found on the one hand .there are some reports showing correlation between volatiles and molecular markers however in other species no correlation was found

which can be explained by considering the fact that the

synthesis of secondary metabolites corresponds to a small portion of the overall information contained in the plant genome.RAPD markers have been used in aromatic plants, with special emphasis in the Lamiaceae (Labiatae). The genus Ocimum is an example where these markers have been used [29] to help understand the genetic relationships. These authors state that RAPD is a sensitive, precise and efficient tool for genomic analysis in this genus. In Ocimum basilicum L. RAPDs have been used together with essential oil composition and agro-morphological characteristics to estimate the relationships among several basil cultivars [30]. The genetic analysis matched the agronomic traits classification however the essential oil profiles produced distinctive clustering. The authors concluded that chemical constituents are not necessarily correlated with taxonomy. The use of AFLP analysis to unravel taxonomy in O. basilicum has confirmed the previous report showing that genomic similarity does not necessarily reflect similarity or difference in output traits, such as oil composition or agronomic traits [31]. These authors state that AFLP dendrograms may be used for universal taxonomic studies, while dendrograms based on end use-related traits, such as oil composition may be of practical interest but do not necessarily correlate with taxonomy. Different results have been obtained in a closely related species, O. gratissimum, L [32]. Cluster analysis using RAPD showed that these molecular markers www.pharmasm.com

IC Value – 4.01

350


ISSN: 0976-7908

Yadav et al

clearly separated three groups which were highly correlated to volatile oil constituents considering the chemotypes. In O. gratissimum the strong correlation found between the molecular markers and the volatiles needs further examination of the actual heritability of each oil constituent, and an F2 population analysis is necessary to determine the actual linkage [32]. In Cunila galioides, RAPDs have been used to discriminate three chemotypes and the authors conclude that two populations from citral and menthene chemotypes are more closely related than the ocimene chemotype which represent a different genetic pool [33] still within the Lamiaceae there was another study using Salvia fruticosa which aimed at establishing the influence of genotype on the chemical profile using 48 Clones represent three populations [34]. It was established in previous study that the patterns of relatedness observed in the chemical profiles corresponded to the genetic profiles generated by RAPD suggesting a genetic basis for the chemical profiles observed. They concluded that the basis of variation in the essential oil composition between clones situated in different climatic regions in the island of Crete was more dependent on the genetic background of the clones than on the climatic variations [34]. On Thymus caespititius, however, using individual accessions, a low correlation was found among essential oil composition and RAPD analysis however molecular data clustered plants according to their geographic origin [35]. RAPD have also been used in understanding the genetic relationships of Origanum × intercedens a hybrid between O. onites and O. Vulgar. The results were compared to those obtained with the essential oil composition and morphological characteristics and it was found that DNA fingerprinting and general morphology placed the hybrid closer to O. onites, while its essential oil showed the hybrid to be more similar to O. vulgare [36]. The authors showed that RAPD markers can be used as reliable tools for the discrimination of the two parental taxa and the putative hybrid in natural populations. In Tanacetum vulgare L. (Asteraceae), a high correlation between the genetic distances based on RAPD patterns and the chemical distance matrices was found, which again reinforces the genetic basis of chemotypes [37]. However, although there are numerous reports claiming a genetic basis to justify the chemotypes, this is not always true. In Leontodon autumnalis (Asteraceae), a phytochemical investigation was conducted using 24 central European populations and www.pharmasm.com

IC Value – 4.01

351


ISSN: 0976-7908

Yadav et al

no correlation was found between the DNA fingerprint profiles obtained using RAPDs and the phytochemical characters such as phenolics and sesquiterpenoids composition [38]. The authors concluded that the morphotypes were of multiple origins or due to different ecological growing conditions rather than genetically determined and that phytochemical races are induced by a limited number of genetic differences which might have occurred independently. In Juniperus Sp. [39] these authors reported that datasets coming from ITS, RAPD and ISSR showed high correlations in contrast to the terpenoid matrix which had a low correlation with all the other matrices. This lack of correlation between data from RAPD analysis and the essential oil composition had been found previously and might be due to a similar composition in the oils [40] .Similar results have been reported more recently on J. brevifolia Azorean individual accessions. Essential oil and molecular analysis using both RAPD and ISSR showed that plants were grouped according to their geographical location not showing identical clustering as with the volatile oil profiles [41]. In peppermint (Mentha × piperita) AFLP analysis was compared to chemotypic composition and the authors concluded that the molecular polymorphisms among the genotypes were most certainly due to many traits in the plant essential oil biosynthesis being one of many functions performed by a plant. However the authors found unique AFLP marker fragments present in only two accessions, with a distinctive chemotype (a high percentage of pulegone and menthofuran). In order to shed light into this question it would be interesting to clone and sequence those AFLP markers for the cluster and to analyze their roles in the essential oil biosynthetic pathway although there is a strong possibility that these polymorphic bands would not have the genetic information for a step in biosynthesis but might rather be another piece of DNA in the vicinity of the gene [42]. In pepper (Capsicum annuum), phylogenetic and mapping studies have been performed using microsatellites (SSRs).Furthermore some of this information can be shared by researchers as there are around 180 pepper SSRs available in the public domain. This increasing availability of genic DNA sequences provides an alternative development for SSR markers since data for ESTs genes and cDNA clones can be downloaded from Gen Bank and compared for the identification of sequences. A recent paper reports the development of a new set of SSR markers from genic Capsicum spp. DNA sequences and describes the potential utility of these assays for the www.pharmasm.com

IC Value – 4.01

352


ISSN: 0976-7908

Yadav et al

development of high-resolution integrated genetic maps in pepper [43]. Also on Origanum, Novak and collaborators reported the use of SSR markers that were developed from expressed sequence tags (ESTs) obtained from the essential oil glands of O. vulgar [44]. These markers can be used as a way to measure genetic variability and heterozygosity but are not related to particular chemotypes. To correctly address the question of finding molecular markers for chemotype or essential oil composition, it seems clear that more specific markers are needed. As an example, in oregano a molecular marker for chemotype formation, which involves the Gaterp marker (γterpinene synthase gene) has been reported, providing a successful approach to understanding the chemotype genetic basis [45]. In other plants that have been more intensively studied, such as rice it was possible to localize in chromosome 8 the gene responsible for fragrance in this species. This trait is recessive and a deletion in that gene will result in a frame shift, causing synthesis of a non-functional enzyme and aroma [46]. In this particular example, it is easy to discriminate between a fragrant and a non-fragrant variety. Genetics and Functional Genomics Plant genomes (the word ‘genome’ refers to the complete genetic make-up of an organism) are variously estimated to contain 20000–60000 genes, and perhaps 15–25% of these genes encode enzymes for secondary metabolism. Clearly, the genome of a given plant species encodes only a small fraction of all the enzymes that would be required to synthesize the entire set of secondary metabolites found throughout the plant kingdom [47]. Molecular techniques are showing continual advances in preparing DNA, sequencing genes, aligning sequences and designing software for interpreting the data. All of this results in vast and numerous independent datasets. This also led to a new area of research called genomics, which can be defined as the study of all the nucleotide sequences including structural genes regulatory sequences and non-coding DNA fragments within all the chromosomes of an organism. Genomic research nowadays has achieved the sequencing of several model and crops species, and the next phase of plant genomics will necessarily build on new phylogenies assisted by molecular techniques, although its interpretation will always be associated with traditional botanical knowledge [48]. The ultimate task will be the determination of the biochemical and cellular function www.pharmasm.com

IC Value – 4.01

353


ISSN: 0976-7908

Yadav et al

of the genes, and transcriptomics plays an important role in this regard because it includes RNA expression profiling. Also genomics walks hand-in-hand with proteomics, which can be understood as protein identification and expression profiling. Concerning secondary metabolism three major questions have to be addressed. (1) What enzymes catalyze the formation of intermediates/final products? (2) What are the genes that encode those enzymes? (3) How are those genes regulated namely which regulatory factors are involved in controlling the individual biosynthetic pathways and ultimately the entire metabolic network? Plant genomics and proteomics provide a large contribution to secondary metabolism research, helping in the identification of enzymes their substrates and products. It is known that a large proportion of the genes in any plant genome encode for enzymes of primary and secondary metabolism. However not all primary metabolites found in all or most species have been identified and only a small proportion of the estimated secondary metabolites have been studied to date. The challenge in the near future will be the elucidation of these metabolic processes. Another important consideration is that the identification of a gene is not necessarily followed by the identification of that gene’s function at the biochemical level. Often, biochemical function is simply inferred from homology to previously investigate sequences, sometimes to a sequence that is several degrees removed. In other cases, when a mutant showing a distinctive phenotype is available the gene function is simply defined as being responsible for or involved in bringing about the structure or developmental process that is defective in the mutant without any specific biochemical function being assigned to the protein encoded by the gene [49]. Concerning aromatic plants and spices the phenotypes are unfortunately quite subtle and difficult to identify because of the volatile nature of the substances involved. State of the Art with Model Plants A valuable contribution to genomics has been the sequencing of whole genomes including Arabidopsis thaliana in 2000 [50] rice (Oryza sativa) and poplar trees (Populus trichocarpa) 2 years later followed by computer analysis of the coding information of the genome and by the analysis of the expression of the entire set of genes by means of DNA microarrays[51,52]. Arabidopsis thaliana contains the smallest plant genome (125 Mb) www.pharmasm.com

IC Value – 4.01

354


ISSN: 0976-7908

Yadav et al

and is therefore the model for studying plant genetics. A mega base (Mb) is a unit of length for DNA fragments equal to 1 million nucleotides. In the human genome 1 Mb is roughly equivalent to one centimorgan (1 cM) or 1 million base pairs (b.p). The poplar genome is roughly the same size around 550 Mb [53]. It is important to realize however that although the entire sequence of the Arabidopsis genome is available [50] the function of many of its genes and sequences are still unknown. In fact nearly 25% of the genes in the fully sequenced and annotated Arabidopsis genome are hypothetical genes with structures that are predicted by computer algorithms while others have already been tested and found to be represented in cDNA preparations [54]. Arabidopsis has been used extensively to study primary metabolism but generally it is not suitable for studying secondary metabolism because the levels of compounds such as volatiles is extremely low. Nevertheless sequence comparisons of genes that encode for enzymes involved in the biosynthesis of secondary metabolites in the Arabidopsis genome often reveal existing related sequences [55]. When a complete genome sequence is not available as is the case for most plant species cDNA libraries and increasingly EST databases have been used as the source of DNA sequence information [49]. Complementary DNA or cDNA is obtained from a mature mRNA template in a reaction catalyzed by reverse transcripts and so cDNAs represent genes that are expressed.ESTs are small DNA sequences (around 200–500 nucleotides) generated by sequencing either one or both ends of an expressed gene (cDNA). These sequenced tags can be used to identify genes expressed in certain cells tissues or organs from different organisms and compare them with the respective genes from CDNA libraries or genome information. ESTs are becoming an important tool to study gene expression and until 2003 about 176915 ESTs had been reported for the Arabidopsis genome [56]. Nowadays large-scale sequencing of ESTs has started for many plant species, including onion (Allium cepa), orange (Citru sinensis) poplar (Populurtrichocarpa) cotton (Gossypum hirsutum), coffee (Coffea arabica), potato (Solanum tuberosum) and sunflower (Heliantus annum). Future Prospects A major priority of aroma and flavor research should be to pursue research on the biochemical pathways leading to aroma biosynthesis, including the identification and characterization of the enzymes and genes controlling these pathways. In fact, many of www.pharmasm.com

IC Value – 4.01

355


ISSN: 0976-7908

Yadav et al

the metabolic pathways are poorly identified or not identified at all. Also the genetics that quantitatively and qualitatively control secondary metabolism leading to such an immense natural variation is still far from understood. Plants are capable of synthesizing a variety of secondary metabolites and presently around 100000 such compounds are known [57]. However, only a small percentage of all plant species have been studied to some extent. Based on the database NAPRALERT, it is estimated that of the approximately 250 000 known plant species, only about 15% have been the subject of some kind of phytochemical study. Nevertheless, the area of functional genomics is increasingly growing, with the genome of several plants becoming known and the information shared through existing databases. Unfortunately, secondary metabolism is species-specific, which makes the genome sequence of model plants such as Arabidopsis of only limited value. The specificity of secondary metabolism is so profound that even two closely related species can have different secondary metabolite profiles. However the early parts of most pathways are common to most plants, and thus homology between genes can be used for strategies to clone genes from other plants. The use of ESTs in combination with functional expression is another approach that could bring important advances in the future. The growing number of plant gene sequences with a known function could result in accumulating data that will result in exponential growth of similar genes identified in other plants. Again, a major bottleneck in this area of functional genomics results from the unknown biochemical pathways involved, involving assays for the enzymes involved in secondary metabolism. It would also be interesting to examine the molecular processes that bring about the variability in secondary metabolism, whether at the level of gene regulation, posttranscriptional regulation or protein evolution. ACKNOWLEDGMENTS The authors wish to express their sincere thanks to the Head, Prof.Madhu Tripathi, Department of Zoology University of Lucknow, for providing valuable support and guidance to frame this review article. Abbreviations: RFLP Restriction fragment length polymorphism; RAPD Random amplified polymorphic DNA; AP-PCR Arbitrarily primed-PCR; DAF DNA amplification fingerprinting; AFLP Amplified fragment length polymorphism; SSR Simple sequence www.pharmasm.com

IC Value – 4.01

356


ISSN: 0976-7908

Yadav et al

repeats; SNP Single nucleotide polymorphism; (ISSR) Inter-sequence simple repeat; (ESTs) Expressed sequence tags; (ITS) internal transcribed spacer region. REFERENCES 1. Croteau R, Kutchan TM and Lewis NG: Biochemistry and Molecular Biology of Plants. American Society of Plant Physiology. Rockville, MD, 2000: 1250-1268 2. Wink M: Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochemistry, 2003; 64: 3 -19 3. Dudareva N, Cseke L, Blanc V and Pichersky M: Evolution of floral scent in Clarkia: novel patterns of S-linalool synthase gene expression in the C. brewery flower. Plant Cell, 1996; 8:1137. 4.

Mimura Y, Inoue T, Minamiyama Y and Kubo N: An SSR-based genetic map of pepper (Capsicum annuum L.) serves as an anchor for the alignment of major pepper maps.Breed Sci. 2012; 62: 93–98

5. Kumar LS: DNA markers in plant improvement: an overview. Biotechnol. Adv. 1999; 17:143-82. 6. Staub JE, Kuhns LJ, May B and Grun P: Stability of potato tuber isozymes under different storage regimes. JAmer Society Hort Sci 1982; 107: 405–408. 7. Mullis K, Faloona F, Scharf S, Saiki R, Horn G and Erlich H: Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction. Cold Spring Harbor Symp. Quant. Biol., 1986; 51:263-273 8. Botstein D, White R L., Skolnick M and Davis RW: Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet, 1980; 32: 314-31 9. Williams JG, Kubelik AR, Livak KJ, Rafalski JA and Tingey SV: DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res., 1990; 25: 6531-5. 10. Welsh J and Mc Clelland M: Fingerprinting Genomic using PCR with arbitrary primer and a matrix of pair wise combinations of primers. Nucleic Acids Res., 1990; 18: 7213.

www.pharmasm.com

IC Value – 4.01

357


ISSN: 0976-7908

Yadav et al

11. Ellsworth DL, Rittenhouse KD and Honeycutt RL: Artifactual variation in randomly amplified polymorphic DNA banding patterns. Biotechniques, 1993; 14: 214-7 12. Jones CJ, Edwards KJ, Castaglione S, Winfield MO, Sala F, van de Wiel C, Bredemeijer G, Vosman B, Matthes M, Daly A, Brettschneider R, Bettini P, Buiatti M, Maestri E, Malcevschi A, Marmiroli N, Aert R, Volckaert G, Rueda J, Linacero R, Vasquez A and Karp A: Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories. Mol. Breed., 1997; 3: 381. 13. Staub J, Bacher J and Poetter K: Sources of Potential Errors in the Application of Random Amplified Polymorphic DNAs in Cucumber Hortscience, 1996; 31: 262-266 14. Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J, Peleman J and Kuiper M: AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res., 1995; 11: 4407-14. 15. Gupta M, Chyi YS, Romero-Severson J and Owen JL: Amplification of DNA markers from evolutionarily diverse genomes using single primers of simplesequence repeats. Appl. Genet., 1994; 89: 998-1006 16. Meyer W, Mitchell TG, Freedman EZ and Vilgays R: Hybridization probes for conventional DNA fingerprinting used as single primers in the polymerase chain reaction to distinguish strains of Cryptococcus neoformans. J. Clin. Microbiol, 1993; 31:2274-80 17. Wu K, Jones R, Dannaeberger L, and Scolnik PA: Inter simple sequence repeat (ISSR) polymorphism and its application in plant breeding Nucleic Acids Res., 1994; 22: 3257–3258. 18. Zietkiewicz E, Rafalski A and Labuda D: Genome fingerprinting by SSRanchored PCR. Genomics 1994; 20: 176–183 19. Fang DQ, Roose M L, Identification of closely related citrus cultivars with intersimple sequence repeats markers Theor. Appl. Genet 1997; 95: 408–417 20. Álvarez I and Wendel JF: Ribosomal ITS sequences and plant phylogenetic inference. Mol. Phylogenet. Evol. 2003; 29: 417–434. www.pharmasm.com

IC Value – 4.01

358


ISSN: 0976-7908

Yadav et al

21. Baldwin BG, Sanderson MJ, Porter JM, Wojciechowski MF, Campbell CS and Donoghue MJ: The ITS region of nuclear ribosomal DNA: A valuable source of evidence on angiosperm phylogeny. Ann.Mo. Bot. Gard. 1995; 82: 247–277. 22. Hershkovitz MA and Zimmer EA: Conservation patterns in angiosperm rDNA ITS2 sequences. Nucleic Acids Res. 1996; 24: 2857–2876. 23. Hughes CE, Eastwood RJ and Bailey CD: From famine to feast Selecting nuclear DNA sequence loci for plant species-level phylogeny reconstruction. Phil. Trans. R. Soc. B 2006; 361: 211-225. 24. Suh Y, Thien L B, Reeve H E, and Zimmer EA: Molecular evolution and phylogenetic implications of internal transcribed spacer sequences of ribosomal DNA in Winteraceae. American Journal of Botany 1993; 80: 1042-1055. 25. Dubouzet JG and Shinoda K: ITS DNA sequence relationships between Lilium concolor Salisb, L. dauricum Ker-Gawl and their putative hybrid, L. maculatum Thunb..Theor Appl Genet 1999; 98: 213-218 26. Litt M and Luty JA: hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Genet., 1989; 44: 397-401 27. Downie SR, Ramanath S, Katz-Downie DS and Llanas E: Molecular biology of aromatic plants and spices. A review Am. J. Bot 1998; 85:563–591. 28. Feng Tu, Stephen R Downie, Yan Yu, Xuemei Zhang, Weiwei Chen, Xingjin He and Shuang Liu: Molecular systematics of Angelica and allied genera (Apiaceae) from the Hengduan Mountains of China based on nrDNA ITSsequences: phylogenetic affinities and biogeographic implications. J. Plant Res 2009; 122: 403–414 29. Singh AP, Dwivedi S, Bharti S, Srivastava A, Singh V and Khanuja SPS: Phylogenetic relationships as in Osmium revealed by RAPD markers. Euphytica, 2004; 136: 11-20 30. Masi LDe, Siviero P, Esposito C, Castaldo D, Siano F and Laratta B: Assessment of agronomic, chemical and genetic variability in common basil (Ocimum basilicum L.) Eur. Food Res. Technol 2006; 223:273–281

www.pharmasm.com

IC Value – 4.01

359


ISSN: 0976-7908

Yadav et al

31. Labra M, Miele M, Ledda B, Grassi F, Mazzei M and Sala F: Studies on preliminary phytochemical constituents and antimicrobial activity of ocimum tenuiflorumleaves. Plant Science., 2004; 167: 725-731 32. Vieira R F, Grayer R J, Paton A, Simon J E, Genetic diversity of Ocimum gratissimum L. based on volatile oil constituents, flavonoids and RAPD markers.Biochem. System. Ecol., 2001; 29: 287-304 33. Fracaro F, Zacaria J and Echeverrigaray S: RAPD based genetic relationships between populations of three chemotypes of Cunila galioides Benth. Biochem. System. Ecol. 2005; 33: 409- 417 34. Skoula M, Hilali-el I and Makris AM: Evaluation of the genetic diversity of Salvia fruticosa mill. Clones using RAPD markers and comparison with essential oil profiles. Biochemical Systematics and Ecology. 1999; 27: 559-568. 35. Trindade H, Costa MM, Lima AS, Pedro LG, Figueiredo AC and Barroso JG: A combined approach using RAPD, ISSR and volatile analysis for the characterization of Thymus caespititius from Flores, Corvo and Graciosa islands (Azores, Portugal). Biochem. Syst. Ecol., 2008; 37: 670-677 36. Gounaris Y, Skoula M, Fournaraki C, Drakakaki G and Makris A: Comparison of essential oils and genetic relationship ofOriganum × intercedens to its parental taxa in the island of Crete. Biochem.Syst. Ecol., 2002; 30: 249-258 37. Keskitalo M, Pehu E and Simon JE: Variation in volatile compounds from tansy (Tanacetum vulgare L.) related to genetic and morphological differences of genotypes. Biochem. System. Ecol., 2001; 29: 267-285. 38. Grass S, Christian Z, Blattner FR and Stuppner H: Comparative molecular and phytochemical investigaton of Leontodon autumnalis (Asteraceae, Lactuceae) populations from Central Europe. Phytochemistry. 2006; 67: 122 – 131. 39. Adams R P, Schwarzbach A and Pandey RN: The Concordance of Terpenoid, ISSR and RAPD markers and ITS sequence data sets among genotypes: An example from Juniperus. BSE 2003; 31:375-387. 40. Adams RP: Systematics of Juniperus section Juniperus based on leaf essential oils and RAPD DNA fingerprinting. Biochem. Syst. Ecol. 2000b; 28: 515-528.

www.pharmasm.com

IC Value – 4.01

360


ISSN: 0976-7908

Yadav et al

41. Lima AS, Trindade H, Figueiredo AC, Barroso JG and Pedro LG: Molecular and volatiles characterization of Portuguese Thymus caespititius chemotypes. Acta Horticulturae, 2010; 860: 81-86 42. Shasany AK, Gupta S, Gupta MK, Singh AK, Naqvi AA and Khanuja SPS: Chemotypic Comparison of AFLP Analyzed Indian Peppermint Germplasm to Selected Peppermint oils of Other Countries J. Essent. Oil Res., 2007; 19: 138145 43. Portis E, Nagy I, Sasvári Z, Stágel A, Barchi L and Lanteri S: Gene-based microsatellite development for mapping and phylogeny studies in eggplant. BMC Genomics, 2008; 9: 357 44. Novak J, Lukas B, Bolzer K, Grausgruber-Grocer S and Degenhardt J: Identification and characterization of simple sequence repeat markers from a glandular Origanum vulgare expressed sequence tag. Molecular Ecology Resources. 2008; 8: 599–601 45. Lukas B, Samuel R, Novak J and Marjoram A :Molecular Marker for Chemotyping in some Important Species of the Genus Origanum 86-4th International Symposium Breeding Research on Medicinal and Aromatic Plants; Ljubljana, SLOVENIA 2009; 17-21, 46. Bradbury LMT, Henry RJ, Jin Q, Reinke RF and Waters DLE: Development of an allele specific amplification (ASA) co-dominant marker for fragrance genotyping of rice cultivars. Mol. Breed., 2005; 16: 279-283 47. Pichersky E and Gang D: Genetics and biochemistry of secondary metabolites in plants an evolutionary perspective. Trends Plant Sci., 2000; 5: 439-445 48. Daly DC, Cameron KM and Stevenson D W: Plant Systematic in the Age of Genomics. Plant Physiol., 2001; 127: 1328-33 49. Fridman E and Pichersky E: Curr. Opin Metabolomics, genomics, proteomics, and the identification of enzymes and their substrates and products Plant Biol.,2005; 8: 242. 50. Arabidopsis Genome Initiative, The Arabidopsis genome initiative. Empirical Analysis of Transcriptional Activity in the Arabidopsis Genome. Nature, 2000; 408: 796-815 www.pharmasm.com

IC Value – 4.01

361


ISSN: 0976-7908

Yadav et al

51. Yu J, Hu S, Wang J, Wong GK-S, Li S, Liu B, Deng Y, Dai L, Zhou Y, Zhang X, Cao M, Liu J, Sun J, Tang J, Chen Y, Huan X, Lin W and Ye C: A Draft Sequence of the Rice Genome (Oryza sativa L. ssp. japonica) Science.

2002;

296: 79-92 52. Goff SA, Ricke D, Lan TH, Presting G, Wang R, Dunn M, Glazebrook J, Sessions A, Oeller P, Varma H, Hadley D, Hutchison D, Martin C, Katagiri F, Lange BM, Moughamer T, Xia Y, Budworth P, Zhong J and Colbert M: A Draft Sequence of the Rice Genome (Oryza sativa L. ssp. indica). Science, 2002; 296: 92-100 53. Wullschleger SD, Jansson S and Taylor G: Genomics and Forest Biology Populus Emerges as the Perennial Favorite. American Society of Plant Biologists Plant Cell, 2002; 14: 2651-2655 54. Reuben S, Cseke L J, Bhinu S, Narasimhan K, Jeyakumar M and Swarup S: In Natural Products from Plants,. Cseke L, Kirakosyan A,Kaufman P B, Warber S L, Duke, JA and Brielmann HL. (eds). CRC Taylor &Francis: Boca Raton, FL, Molecular biology of plant natural products; 2006; 632. 55. Facchini PJ, Bird DA and St-Pierre B: Can Arabidopsis make complex alkaloids. Trends Plant Sci., 2004; 9: 116-122 56. Zhu W, Schlueter SD and Brendel V: Refined Annotation of the Arabidopsis Genome by Complete Expressed Sequence Tag Mapping. Plant Physiol., 2003; 132: 469-84 57. Verpoorte R, Vander Heijen R and Memelink J: Engineering the plant cell factory for secondary metabolite production. Transgenic Res, 2000; 9: 323–343 For Correspondence: Shiv S Yadav Email: [email protected]

www.pharmasm.com

IC Value – 4.01

362