The use of molecular markers in the last few decades for the genetic analysis has become best choice in plant biotechnology and molecular genetics. Mainly there are two types of markers; those which are developed without prior sequence information and those which are site-targeted and designed on the basis of sequence information. These techniques have various principles, procedures and limitation and choice of marker depends on objectives need to be completed by a researcher. They are applicable in various fields such as genetic diversity, genomic fingerprinting and mapping, population genetics, taxonomic studies and plant breeding programs.
Introduction
A molecular marker is one of the significant approaches for the evaluation of genetic polymorphism. Molecular markers have been extensively used for the identification and authentication of plant taxa. These markers are not influenced by age, physiological condition of samples and environmental factors (Yip et al., 2007). Molecular markers are not tissue-specific and thus can be detected at any developmental stage of an organism. On the basis of their utility in various fields of life sciences, molecular markers can be divided into three types,
Visually assessable traits (Morphological and Agronomic traits/markers).
Markers based on gene product (Biochemical Markers).
Those relying on a DNA assay (Molecular Markers).
For long time, species were identified on the basis of morphological traits. However, it has well been documented that different individuals of same species have dissimilarity in their morphology which could be due to local environmental factors and create taxonomic controversies (Duminil and Michele 2009). Cryptic species which are morphological identical but have a large variation at molecular level. However it is difficult to make differentiation among cryptic species on the basis of morphological characters. Moreover, on the basis of morphological characters it is difficult to have access to the vegetative part of adult woody individuals, especially in tropical forest ecosystem (Duminil et al., 2006).
The use of biochemical markers including seed storage proteins and isozymes is a comparatively powerful method for determination of allelic frequencies of specific genes. Although these types of markers provide better information about population subdivision and are also useful for determination of genetic variability. But there are some limitations including the few numbers of polymorphic enzymatic systems and failed to depict the variability of whole genome. Proteins markers are also influenced by methodology, tissue type and developmental stage of plants (Linda et al., 2009).
The efficiency of DNA-based markers is so high to discriminate closely related varieties and even individuals of same species. They have proved their utility in various fields such as genetic diversity, genomic fingerprinting and mapping, population genetics, taxonomic studies and plant breeding programs. DNA markers have specific location on a genome and should not be considered a gene, because they usually have no specific biological function. The genetic markers have difference with respect of copy number, polymorphic profile, locus specificity, level of reproducibility, technical requirement and financial value (Kumar et al., 2009).
A diverse range of DNA-based marker technologies has been established and they can classified in two three broad types, namely polymerase chain reaction (PCR)-based markers, hybridization-based markers and sequencing-based markers. Genetic or DNA based marker techniques such as Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD), Simple Sequence Repeats (SSR) and Amplified Fragment Length Polymorphism (AFLP) are now in frequent use in various fields of plant studies and crop improvement programs on commercial level (Ayed et al., 1995; Semagn et al., 2006; Kumar et al., 2009; Primmer, 2009). The objectives of present studies are to discuss types of molecular markers and their basic principles with application in various fields of plant biotechnology.
PCR-based markers:
Amplification of a particular DNA segments or loci, enzymatically with the help of various techniques based on the use of primer either specific or random in nature (Scott et al., 1993). There are some advantages of PCR-based methods over hybridization-based methods including small amount of DNA is used and revealed high polymorphism within a short time. There are basically two types of PCR-based techniques, depending on the characteristic of primers used for amplification.
Arbitrary or semi-arbitrary primer PCR techniques that developed without former sequence information (e.g., AP-PCR, DAF, RAPD, AFLP and ISSR). Generate high polymorphism that enables to develop many genetic markers (Semagn et al., 2006).
Site-targeted PCR techniques that develop from already reported DNA sequence information (e.g. EST, CAPS, SSR, SCAR, STS).
Arbitrary and Semi-Arbitrary Molecular Markers:
Random Amplified Polymorphic DNA (RAPD):
RAPD primers which are mostly 10bp arbitrary sequence was first developed by Welsh and McClelland in 1991 (Jones et al., 1997). The main advantage of RAPD is that they are quick and easy to assay. RAPD primers are randomly dispersed throughout the entire genome and have a very high genomic abundance (Kumar et al., 2009). Usually, 5-50ng/ul quantity of DNA is needed for one reaction and these primers re commercially available in market and easy to access. RAPD markers are condemned because of their low level of reproducibility (Schierwater and Ender 1993), and hence high standardized experimental procedures are required because of their sensitivity to the reaction conditions. Several factors are involved to affect the reproducibility of RAPD reaction: quality and quantity of DNA, concentration of magnesium chlorides and PCR conditions specifically annealing temperature of primers and extension time (Wolff et al., 1993; Semagn et al., 2006).
RPAD markers have been used to reveal diversity within and among species (Esselman et al., 2000). Random primers have also been applied in gene mapping (Willains et al., 1990; Hadrys et al., 1992). RAPD, AP-PCR (arbitrarily primed PCR) and DAF (DNA amplification fingerprinting) have placed in a group named as MAAP (Multiple arbitrary amplicon profiling) (Caetano-Annolles, 1994). AP-PCR primers are larger arbitrary primers than RAPDs. While DAF markers are 5-8bp primers and used to generate larger number of fragments (Kumar et al., 2009).
Amplified fragment length polymorphism (AFLP):
AFLP technique is a combination of RFLP and PCR procedures (Lynch and Walsh, 1998). First, genomic DNA is digested with restriction enzymes and then digested DNA with ligated adopters are preamplified followed by selective amplification by using primers anneal to adopters and restricted sites (Vos et al., 1995). AFLP fingerprinting is a new and powerful technique to determine genetic diversity of any organism and generate AFLP markers without development of primer from sequence database (Semagn et al., 2006).
AFLP banding patterns are visualized on either agarose or polyacrylamide gels. Polyacrylamide gel electrophoresis (PAGE) provide maximum resolution of AFLP fragments up to the extent of single-nucleotide length differences, whereas it is difficult to score fragment size difference of less than ten nucleotide on agarose gels.
An AFLP technique has potential to revealed high polymorphism as compared to other techniques like RFLP and RAPD (Mueller et al., 1996; Lin et al., 1996; Powell et al., 1996; Jones et al., 1997). This technique is also applicable in phylogenetic studies of closely related species, to identify cultivars, and in the gene mapping (Vos et al., 1995; Herselman, 2003). Although AFLP genetic markers have strength to produce a large number of polymorphic bands but the major drawback is that these markers are dominant and cannot differentiate homozygotes and heterozygotes. According to Percifield et al. (2008) worked on Hypericum a medicinally important genus and reported that AFLP markers not only used to estimate relationship in accession but also used in authentication of material. Moreover, RNA fingerprinting through Cdna-AFLP techniques had used to screen genes differentially expressed under stress condition (Rodriguez et al., 2005). Ch et al., (2003) studied on wheat lines under salt stress condition and found a large number of genes which are specifically related to salt stress condition.
Inter Simple Sequence Repeat (ISSR):
ISSR is a broad term for a genome segment between microsatellite loci and first time available as molecular markers in 1994 (Zietkiewicz et al., 1994). ISSR are semi arbitrary markers amplified by using one primer complementary to target microsatellites and primer having core sequence of microsatellite along with few selective nucleotides. ISSR technique does not need genome sequence information for primer designing. These markers produce multilocus and highly polymorphic banding pattern which making it ideal for RFLP and RAPD techniques (Zietkiewicz et al., 1994; Tsumara et al., 1996; Nagaoka et al., 1997). ISSR markers are rapid and easy to handle, but unlike RAPD markers they have potential of high reproducibility because of longer length of their primers (16-18bp). ISSR usually segregate as dominant markers (Gupta et al., 1994; Tsumara et al., 1996; Ratnaparkhe et al., 1998; Wang et al., 1998), but there are also reports of codominant segregation (Wu et al., 1994; Akagi et al., 1996; Wang et al., 1998; Sankar and Moore 2001). ISSR markers have been used to study genetic characterization of phytoplankton (Bornet et al., 2004). In higher plants, particularly ISSR markers are in demands, because they are known to be highly informative, and quick to use (Zietkiewicz et al., 1994; Bornet and Branchard 2001)
Sequencing-based based-PCR-markers
Sequencing based PCR markers are used to detect variation at a single nucleotide level due to transversion, insertion or deletion. Through sequencing can efficiently identify such single nucleotide polymorphisms that usually used to compare the closely related organisms
Simple Sequence Repeats (SSRs):
Microsatellites, simple sequence repeats (SSRs) are repeating sequences of 1-6 base pairs of DNA of most eukaryotic species (Bidich and Anidal, 1998). SSR markers revealed high level of inter- and intra-specific polymorphism, particularly when tandem repeats number ten or greater (Bown and Wheals, 2006). These repeated sequences are simple and often consist of two, three or four nucleotides, and can be repeated 10 to100 times. As compared to other molecular markers, SSRs could a better choice because of their high reproducibility, high copy number, codominant segregation, genomic abundance and random distribution through entire genome and widely applicable in Plant breeding and genetics in last few decades. Moreover, low quantity and quality of DNA is required because of long PCR primers (Morgante et al., 2002).
Primers for SSRs technique can be designed either by constructing genomic library or by screening sequences from reported (Semagn et al., 2006). Sequence information generated from various means including EST sequencing projects, characterized genes and from Cdna clones of various plant species are deposited in online databases and are referred as EST-SSRs and genic microsatellites respectively. EST-SSRs and genic microsatellites are simple and economical as compared to genomic microsatellites because their sequence can easily be retrieved from databases which are publically accessible (Varshney et al., 2005).
More than 6 million ESTs are now available in computerized data. EST-SSR markers can be used for direct allele selection, if they are shown to be completely linked or even responsible for targeted trait (Sorrells and Willson 1997). In wheat EST-SSR markers had identified associated with photoperiod genes further utilized in genomic map construction in several plants (Yu et al., 2004; Nicot et al., 2004; Holton et al., 2002; Gao et al., 2004). The limitation of using SSRs is genic microsatellites are developed for only those species of which sequence is available in databases whereas, high development costs are involved if adequate primer sequences for the species of interest are unavailable, thus making them difficult to apply to unstudied groups (Kumar et al., 2009).
These markers are highly informative and can even be used in identification of clones and strains and considered ideal in gene mapping (Hearne et al., 1992; Morgante and Olivieri 1993; Jarne and Lagoda 1996). Recently SSRs markers are widely used to analyze genetic diversity crop plants including Alfalfa population (Falahati-Anbaran et al., 2007), Jute cultivars (Akter et al., 2008), wheat cultivars (Salem et al., 2008) and popcorn genotypes (Leal et al., 2010).
Sequence Characterized Amplified Region (SCAR):
SCARs markers are specifically used to amplify specific locus and was introduce by Michelmore et al., and Martin et al. (1991). In this technique amplified segment by RAPD markers linked to a specific trait, is sequenced and primers of 15-30bp long are developed. The major advantage of this technique is that SCARs are locus specific and is useful to develop species-specific markers (Scheel et al., 2003). Dwivedi et al. (2007) reported SCARs sequences linked only to apomictic process. Similarly, SCAR marker associated with disease resistant gene Rpf1 (Red Phytophtora fragariae) was developed to screen resistant varieties of strawberry. SCARs are mostly used in gene mapping studies and marker assisted selection (Paran and Michelmore 1993). Yamashita et al. (2005) develop SCAR markers linked to Rf (fertility restoring gene) in Allium fistulosum. The SCAR markers OPJ15700 is reliable for identification of male fertile and male sterile plants. Similarly, SCARs linked to ben gene (bentazon susceptible lethality gene) were used to identify the homozygous and heterozygous genotypes in to rice cultivar (Tai-he et al., 2003). SCARs have utility in the field of crop breeding and molecular systematic.
Cleaved Amplified Polymorphic Sequence (CAPS):
CAPS technique has also been referred as PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) as it is a combination of PCR and RFLP (Maeda et al., 1990). In this technique amplified product is cleaved by restriction enzymes and variation in restriction sites is visualized on gel electrophoresis (Kumar et al., 2009). CAPS markers are codominant in nature and have been predominantly used in gene mapping studies (Koneczny and Ausubel 1993). CAPS is restricted because fragment size (300-1800bp) is limited and it would be more difficult to find polymorphism. In addition, for primer synthesis sequence information is needed.
Single Nucleotide Polymorphism (SNP):
SNPs markers system is used to detect polymorphism as a result of change in a single nucleotide. SNPs are biallelic markers and thousands of SNPs can be analyzed simultaneously through DNA microarrays. There are several classes of SNPs accordingly their position on the genome. SNPs when present in a gene are named as exon intron and promoter SNPs according to their respective position (Khlestkina and Salina 2006). SNPs are increasingly becoming the marker of choice in genetic analysis and are used routinely as markers in agriculture breeding programs (Gupta et al., 2001). SNPs markers have been used in plants for many molecular genetic marker applications including high-resolution genetic map construction, linkage disequilibrium based association mapping, genetic diagnostic, genetic diversity analysis, cultivar identification, phylogenetic analysis and characterization of genetic resources (Rafalski 2002a).
Single-Strand Conformation Polymorphism (SSCP):
SSCP-DNA based molecular marker type was first introduced in 1989 as a new source of detecting genetic variation. The technique is based on the fact that the electrophoretic separation of single strand DNA depends on the secondary structure (conformation) of the molecule, which is changed significantly with differences in a single locus in sequence (David et al., 2008). Under optimal conditions through SSCP technique it is possible to detect 80-90% potential nucleotide variations. However, sensitivity of SSCPs is affected by temperature and Ph (Wanger, 2002). In SSCPs, DNA fragments of 200-800bp is first amplified by using specific primer of 20-25bp and then denatured to a single stranded DNA. David et al. (2008) supported the use of SSCP based molecular markers in the genotyping study in comparison to microsatellites which would be difficult and expensive due to sequence analysis.
Ribosomal DNA Sequences:
Ribosomal DNA Rdna sequences are mostly used nuclear molecular markers for phylogenetic studies in many angiospermic families. Rapidly evolving 18S-25S internally transcribed spacer (ITS) of ribosomal DNA are widely used as markers in molecular systematic (Hershkovitz et al 1999).
Ribosomal DNA (rDNA) loci, i.e., nucleolar organiser (Nor) and 5S loci, are examples of ribosomal DNA markers. The Nor is a complex genetic locus (found at more than one chromosomal locations) consisting of several tandemly arranged copies of ribosomal RNA (rRNA) genes (Rogers and Bendich, 1987). The basic organisation of rDNA has been preserved in most eukaryotic organisms. Each repeating unit of rDNA consists of conserved coding regions (18S, 5.8S and 25S), and the corresponding variable portion such as internal transcribed spacers (ITS) and an intergenic spacer (IGS).
Indeed, IGSs frequently show adequate variation to analyze the genetic relationships between closely related species, populations or cultivated varieties (Polanco and Perez de la Vega, 1995, 1997; Penteado et al., 1996; Nickrent and Patrick, 1998). They can vary widely in length between plant species groups (ranging from approximately 1 kb to over 12 kb) (Rogers and Bendich, 1987) and within species (Polanco and Perez de la Vega, 1995; 1997; Penteado et al., 1996), mainly due to the presence of one or more tandem or dispersed subrepeat sequences. As in most eukaryotic species, the 5S rDNA of plant species is organized in tandem repetitive units located at one or more loci. Each repetitive unit includes 120 bp coding for the 5S rRNA, and a non-transcribed spacer (NTS) which typically ranges between 100 and 900 bp in length (Sastri et al., 1992). The tandem organization and high copy number of two ribosomal genes(45S Rdna and 5S Rdna) together provide functional markers for chromosome identification and karyotyping in various plant genera including Aegilops (Badaeva et al., 1996), Arabidopsis (Fransz et al., 1998), Hordeum (Takeda et al., 1999) nd various plant species (Maluszynska, 2002).
Chloroplast Genome Sequences
Chloroplast DNA (cpDNA) has been extensively used in the evolutionary studies of various groups of plants. In chloroplast DNA slow rate of evolution and conserved nucleotide substitution make it ideal for the phylogenetic study at different taxonomic level of plants. The ribulose bisphosphate carboxylase gene (rbcL) was one of first chloroplast molecular marker has been widely sequenced from numerous plant taxa for phylogenetic analysis including Cornaceae (Xiang et al. 1993), the Cupressaceae (Gadek and Quinn 1993), the Ericaceae (Kron and Chase 1993), the Geraniaceae (Price and Palmer 1993), the Onagraceae (Conti et al. 1993), and the Saxifragaceae (Soltis et al. 1993). Moreover rbcL gene is sometimes too conserved to discriminate at the generic level as in the tribe Triticeae of the Poaceae (Doebley et al. 1990). Analysis of noncoding regions of cpDNA for phylogenetic studies can be valuable below the family level. (Curtis and Clegg 1984; Wolfe et al. 1987; Zurawski and Clegg 1987; Clegg and Zurawski, 1991).
Interest in chloroplast microsatellites (cpSSRs) is continuously increasing. As cpSSRs are not only analyzed to estimate the cpDNA polymorphism and to study the phylogenetic relationships, but also used as plastome markers of individual cultivars. Chloroplast microsatellites are analogous to nuclear microsatellites, differing from these in being mononucleotide repeats. Yet cpSSRs preserve the main advantages of nuclear microsatellite markers, such as hypervariability and codominant inheritance (Provan et al., 2001). Another way in analyzing cpDNA is the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This was used for the identification of intra-specific variations in the cpDNA (Rieseberg et al., 1992; Tsumura et al., 1995). The chloroplast genes for phylogenetic analysis at the high taxonomic level include matK (Johnson et al., 1994; Hilu et al., 2003), atpB (Wolf, 1997; Soltis et al., 1999), rpoC1 (Samigullin et al., 1999), ndhF (Alverson et al., 1999), rps4 (Buck et al., 2000; Korall et al., 2006) and some others.
Transposable Elements-Based Molecular Markers:
Transposon insertions can have deleterious effects on host genomes, but in the past few decades they have also been exploited as molecular markers. These mobile elements, 60 year ago were first reported in maize. On the basis of potential properties of these elements are broadly classified in to two catagories;
Class I: Retrotransposons, long interspread nuclear elements and encoded Mrna. During transposition event a new copy of the transposon is produced, whereas original copy remains intact at the original site.
Class II: DNA transposons, this type of transposons excise themselves from donor site and integrate themselves at the acceptor site (Grzebelus, 2006).
Retrotransposons (RTN) are characterized as long defined and conserved sequences, provide best opportunity for the development of marker system to be use alone or in combination with other markers (Kalendar et al., 1999). These markers relies on amplification using a primer construct by using sequence of retrotransposons and a part of the adjoining segment of genome . In this set of molecular markers include ; sequence-specific Amplified Polymorphism (S-SAP), Inter-Retrotranposon Amplified Polymorphism (IRAP), Retrotranposon-Microsatellites Amplified Polymorphism (REMAP), Retrotranposon-Based Amplified Polymorphism (RBIP) and finally, Transposable Display (TD). RTN-based markers are especially suitable for the study of genetic diversity and phylogeny and have been applied in the genera such s Brassica (Totout et al., 1999), Hordeum (Waugh et al., 1997; Kalender et al., 1999; Kalender et al., 2000), Oryza (Iwamoto et al., 1999; Motohashi et al., 1997), Pisum (Vershinin et al., 1999; Ellis et al., 1998; Pearce et al., 2000).
Applications of Molecular Markers;
Molecular markers in herbal drug technology:
DNA-based techniques have been widely used in pharmacognosy includes all the aspects of drug development and authentication, where biotechnology-driven applications play an important role (Joshi et al., 2004). DNA-based markers have been widely used for authentication, hereditary variation/genotyping, detection of adulteration/substitution, selection of desirable chemotypes, plant breeding of plant species of medicinal importance. Most of the medicinally important plants species are adulterated with other morphologically similar species or varieties. RAPD markers clearly differentiated dried fruit samples of Lycium barbarum from its related species (Zhang et al., 2001). Panax ginseng which is medicinally important and has antioxidant and anticancer effect was discriminate among ginseng population with the help of RAPD and PCRâ€"RFLP (Um et al., 2001). Geographical conditions affect the medicinally active constituents and hence through RAPD markers different accession Taxus wallichiana (Shasany et al., 1999), neem (Farooqui et al., 1998), Juniperus communis L. (Adams et al., 2002b), Codonopsis pilosula (Fu et al., 1999), Allium schoenoprasum L. (Friesen et al., 1999), Andrographis paniculata (Padmesh et al., 1999) were differentiated. SCAR markers were developed for Phyllanthus amarus, P. debilis and P. urinaria (Theerakulpisut et al., 2008). Inter- and intra-species variation has also been studied in different genera such as Glycerrhiza (Yamazaki et al., 1994), Echinacea (Kapteyn et al., 2002), Curcuma (Chen et al., 1999) and Arabidopsis (Lind-Hallden et al., 2002) by using RFLP and RAPD markers.
Authentication of Taraxacum mongolicum (herba taraxaci) was done successfully by using AP-PCR and RAPD. Chloroplast trnL/trnF sequence were used for identification of medicinal rhubarb, which is absent in its adulterants (Yang et al., 2002). Similarly, DNA sequence analysis of rDNA ITS and PCRâ€"RFLP were identified for their utility in identification of four medicinal Codonopsis species from their related adulterants, Campanumoea javania and Platycodon grandiflorus. The technique is effective and reliable for differentiation of Codonopsis from the adulterants (Farooqui et al., 1998).
DNA-microarray technology has also proved utility in herbal research and development. For microarray genotyping it is necessary to have distinct DNA sequence of medicinally important species. This DNA sequence information is further used to develop probe on a silicon-based gene chip. These probes are used for authentication of material in the test sample being analyzed (Chavan et al., 2006).
Recently, molecular marker technology has applied in the field of neutraceuatical. Transgenic approaches have also been made to improve the yield and quantity of natural plants products through identification of specific genes (Kumar and Gupta 2008). Primers specific for inserted genes in Roundup ReadyTM soybean have been found to be suitable for detection and discrimination of GM soybean from non-GM products (Lin et al., 2002). In another study, Roundup Ready soybeans, Bt 176 maize and Cecropin D capsicum have been successfully discriminated from non-GM products using primers specific for inserted genes and crop endogenous genes (Deng et al., 2002).
According to the new European Council legislation, the labeling of food products containing licensed GMO must specify the percentage of amount, where they are present at or above a level of 1%. To fulfill the council labeling regulation for GM foods, PCR methods were developed for identification in several countries of Europe and for quantitative real-time PCR analysis was undertaken (http://europa.eu.int/eur-lex/en/lif/dat/2000/en_300R0049.html).
Molecular Markers and Plant Improvement:
Identification of plants or progenies containing the desired gene combinations for the trait of interest is one of the most difficult problems in plant breeding. In plant breeding programs, it is vital to have sufficient genetic diversity for the production of new varieties so that plants can tolerate biotic and abiotic stress. Molecular markers act as a tool in the identification of major gene, QTLs, or to introduce new characters in elite germplasm. In wheat, molecular markers have been used to identify forty traits of economic importance (Gurta et al., 1999).
Marker assisted selection (MAS) is a program which making it easier for scientists to select plant traits and develop new variety. MAS involve simply laboratory assays that can be more accurate and less expensive than tradition trait assay. Breeders have screened number of soybean (Glycine max) fungal diseases in conjunction with SCN (soybean cyst nematodes). With the help of molecular markers disease resistant soybean varieties have been developed (Webb 2000; Webb et al. 1995). In the USA, many companies use MAS to select for SCN varieties (Chill and Schmidt 2004).
Resistance to biotic and abiotic stess is one of the most important targets during the improvement of crops. Fujimori et al. (2004) studies resistant to crown rust in Italian rye grass and identified that DNA markers (AFLP in this case) are tightly linked to resistant genes and provide new strategies for development of new varieties. Similarly, resistant genotypes have also been developed against Barley yellow mosaic virus (ref…….) in Barley and resistance to Fusarium head blight in wheat.
Abiotic stress, especially temperature and drought stress are the primary cause of plant loss worldwide. The quantitative trait loci (QTL) mapping is one excellent approach to solve issues related to plant stress tolerance. The response of plants to abiotic stress is multigenic in nature. QTLs related with abiotic stress tolerance have been screened in many significant crop species, salt tolerance in rice, drought stress in cotton and cold stress in woody plant salix. Molecular markers also used to differentiate stress tolerance and stress susceptible plants.
Identification or fingerprinting:
DNA markers have been used for identification or fingerprinting purposes of both individuals and populations especially those detected by multilocus probes. In individual identification DNA markers can assist in selecting the genome type for purpose of germplasm conservation in order to include the widest genetic variation possible. DNA markers are well suited for the determination of genetic relatedness. The basic idea in these studies is to characterize genome diversity of plants. Dendrograme from different genotypes based on cluster analysis (UPGMA) of genetic distance from DNA markers indicated that molecular markers are suited to measure the average level of relatedness and to identify closely related genotypes.
Both ISSR and AFLP can be applied for the identification of large number of Popular accession, and can also be used to rapidly determine the genetic relationship among them (Jianming et al., 2006). Tobacco (Nicotiana spp.) is one of the most important commercial crops in the world. Zhang et al., 2005 employed RAPD techniques to assess the polymorphism, similarities and relationships among N. tabacum L. cultivars. It was also investigated that RAPD assay could discriminate flue-cure Virginia (FCV) tobacco and burley tobacco cultivars (Saraka and Rao 2008). RAPD markers have also proved to be more efficient than standard morphological markers for the identification of apple rootstocks (Koc et al., 2009). As it is well known, rootstocks influence several aspects of fruit tree growth and development, including yield and fruit quality (Webster 1995, Filho et al., 2007).
Novakova et al., 2009 analyzed potato varieties using PCR-IRAP (Inter-Retrotransposan Amplified Polymorphism) method in order to distinguish unambiguously the varieties. Moreover, microsatellites are suggested as the suitable markers for the identification of different Trifolium repens cultivars. RAPD markers have also revealed high genetic variation within the cultivars (Dolanska and Curn 2004). Phylanthus amarus is a medicinal plant and was being used in traditional medicines. Sequence-Characterized Amplified Regions (SCARs) were designed from nucleotide sequence of specified RAPD markers for species specification of P. amarus and produced useful results (Theerakulpisut et al., 2008). DNA fingerprinting using specific SSR markers segregated cultivars of two jute species (Corchorus olitorius and Corchorus capsularis) and provided unique genetic identities of various jute cultivars (Akter et al., 2008). Brassica is the second most important oil seed crop in the international oil seed market after Soybean (Christopher et al., 2005). To improve yield and quality of oil content of Brassica , presence of sufficient genetic diversity in the germplasm is an important prerequisite. RAPD and SSR markers showed high genetic dissimilarity and results can be used infuture Brassica breeding programe (Abbas et al., 2009). Similarly ISSR markers were used to assess the identification of 23 important Ficus species/varieties and determination of genetic relationship among these species. Both molecular and morphological markers will be helpful in the preservation of the Ficus germplasm which is one of the varieties/accessions facing genetic erosion (Rout and Aparajita 2009).
Molecular markers in threatened plant species:
The genomic study of threatened species has been of great concern to both evolutionary study and for planning conservation strategies for long time (Avis and Hamrick 1996, Young and Clarke 2000 and Hedrick 2001). To preserve the evolutionary history of threatened species, it is important to have knowledge on the genetic makeup of species (Godt and Hamrick 1998). Genetic diversity of rare and endangered species is subjected to strong random changes in allele frequencies called genetic drift. Genetic drift can cause the loss of alleles from the population and ultimately the loss of polymorphism such that a locus becomes limited to a single allele (Lowe et al., 2004). Thus, molecular markers can be valuable approach for investigating the pattern of genetic diversity in threatened species, and clarifying demographic and ecological issues early in species management in order to plan long term conservation and restoration projects (Kim et al., 2005). A low level of genetic variability in changing environment often result in minor fitness of individuals (Oostermeijer et al., 1994; Fisher and Mathies 1998; Luijten et al., 2000; Hansson and Westerberg 2002) and in extreme cases causes the extinction of species (Young et al., 1996). However, some endemic species exhibit high level of genetic diversity contrast to their congeners (Torres et al., 2003; Conte et al., 2004; Ellis et al., 2006). Helianthus verticillatus is an extremely rare species of sunflowers, which is reported from only three locations in North America. H. verticillatus showed significantly higher level of genetic diversity using nuclear and microsatellites than the common H. angustifolius (Ellis et al., 2006).
Limonium cavanillessi is an extremely endangered plant species endemic to the east Miditerranean region of Spain. Palacios and Gonzalez-Candelas (1997) analyzed genetic variation by using RAPD markers and detected low genetic variation in plants. Whereas, Caralluma genus is an important medicinal and threaten succulent plants in Pakistan. High level of genetic variation was observed by using RAPD markers and results can be used for the conservation of Caralluma in Pakistan (Mahmood et al., 2009).
Adiantum reniforme var. sinense has low reproductive ability and stringent habitat requirements, which cause it to be rare and endangered (Lin 1980). Liu et al., 2007 observed low genetic variation through RAPD markers and considered it to be one factor causing endangered status of this taxon.
