PCR-based Markers: A Review Pareek Nisha1, Grover Staffi2, Singh Yaksha3, Malik CP4,* 1Ph.D. Students, School of Life Sciences, Jaipur National University, Jaipur, Rajasthan, India 2Ph.D. Students, School of Life Sciences, Jaipur National University, Jaipur, Rajasthan, India 3Ph.D. Students, School of Life Sciences, Jaipur National University, Jaipur, Rajasthan, India 4Advisor (Academics), School of Life Sciences, Jaipur National University, Jaipur, Rajasthan, India *Email id: cpm_malik@yahoo.com
Abstract Markers are becoming resourceful tools in assessing genetic diversity among genotypes. Detection and analysis of genetic variation plays an important role in understanding the molecular basis of various biological phenomena in biota and in establishing phylogenetic relationships among cultivars. A marker can be a protein molecule or a nucleotide sequence linked with a particular trait of an individual and can be employed to predict or characterise genotypic differences. Markers can be morphological, biochemical, cytological and DNA based. DNA-based markers offer several advantages over the conventional markers (morphological, biochemical, cytological) as they are independent of environmental factors, innumerable, highly polymorphic, reliable and lack pleiotropic or epistatic effects. Information provided by them can be analysed objectively; giving new direction to plant breeding programs. DNA based markers are categorised into non-PCR-based and PCR-based. Discovery, principle and applications of PCR-based markers are discussed. Top Keywords DNA based Markers, Pleiotropic, Epistatic, QTLs. Top |
Introduction Variability among individuals arises due to polymorphism at the DNA level. Knowledge of genetic diversity among organisms is important in drawing an appropriate method for their survival, adaptability, selective breeding, rapid domestication and conservation. Analysis of genotypic differences within and among the populations can be performed by using molecular genetic markers. A marker can be a protein molecule or a DNA segment that is situated near each other on a chromosome and tend to be inherited together. Markers are used in tracking the inheritance pattern of a gene that has not yet been identified, but whose approximate location is known. The availability of a large number of molecular markers and differences in their principles and methodologies requires a good deal of knowledge in choosing one or more of such marker types. The choice of a marker depends on the number of gene loci to be studied, the levels of polymorphism to be analysed and/or the cost to be involved in it. This review deals with the basic principle, advantages, disadvantages and applications of various PCR-based markers in different fields. |
Top Conventional Marker Systems Comprised Morphological (Phenotypic) Markers Any observable trait that can be measured as a difference between any two individuals for example plant height, seed shape, flower colour, etc., serves as a morphological marker. These genetic markers are known to biologists since ancient times. They are easy to spot and are based on disparity in physical appearance. Morphological traits are significant in identification of varieties and in-plant breeding programmes. Phenotypic categorisation provides a partial knowledge of the average functional variant of genes carried by a given species or population, it is cheap but often requires large tracts of land to carry out the experiments, making it more expensive. Moreover these markers are insufficiently polymorphic and are mostly dominant. The traits observed are often liable to phenotypic plasticity, they are few in number and different individuals of the same species may present dissimilarity in their phenotype either naturally or in connection with the local environment. Biochemical Proteins (Isozymes and Allozymes) have more advantages as genetic markers compared to phenotypic markers for they are co-dominant, less influenced by the environment and their development is much easier. Isozymes refer to multiple molecular forms of an enzyme sharing a catalytic activity derived from the tissue of a single organism (Markert and Moller, 1959) and isozymes that are encoded by orthologous genes are called as allozymes. They differ by one or more amino acids due to allelic differences. They originate through alterations in the amino acid sequences, which alters the net electric charge of the protein thereby changing the overall shape (conformation) of the molecule thus affecting the migration rate of proteins during electrophoresis and resulting in their separation into specific banding patterns. Isozymes have been successfully used in many crop improvement programs. They are quick and easy to use; do not require DNA extraction or the availability of sequence information, primers or probes. However they can't be used to construct a complete genetic map as only a limited number of enzymes are available and thus, the resolution of diversity is limited, requiring special staining methods. Cytological Marker These are based on the polymorphisms of number and structure of chromosomes among different species. These can be used as genetic markers to locate other genes on to chromosomes and determine their relative positions, or they are used for genetic mapping via chromosome manipulations such as chromosome substitution (Xu, 2010). They are less influenced by the environment and are mostly used in genetic counselling. Development of cytological markers is labour intensive and time consuming. Moreover, some species are vulnerable to the changes in chromosome number and structure, thereby limiting their wide scale use. Top Modern Markers Desirable Properties of DNA Markers Highly polymorphic and reproducible Easily available, fast and cheap to detect Easy and quick assay Co-dominant inheritance (determination of homozygous and heterozygous states of diploid organisms) and recurrent occurrence in genome Multi-allelic and non-epistatic Selectively insensitive to environmental conditions or management practices and Exchange of data between different laboratories should be easy
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Non-PCR-based (Hybridisation-based) Restriction fragment length polymorphism (RFLP) (Botstein et al., 1980) It involves enzymatic digestion of genomic DNA, followed by separation of restricted DNA fragments by gel electrophoresis and then blotting on to a nitrocellulose membrane. Banding patterns are visualised by hybridising a chemically labelled DNA probe. Variations in the length of DNA fragments produced by a specific restriction endonuclease from genomic DNAs of two or more individuals of a species give rise to polymorphism. The RFLP markers are highly polymorphic, reproducible, show co-dominant inheritance and are efficient in detection of large number of loci. The technique is not used widely because it involves expensive radioactive and toxic reagents, large quantity of high quality genomic DNA and is time consuming. The requirement of prior sequence information for probe generation increases the complexity of the methodology. These drawbacks led to the development of a new set of methods that are technically less complicated like PCR-based techniques. PCR-based Markers Recent advances in the field of plant molecular genetics has led to the development of a number of PCR-based DNA markers. They are relatively simple, quick, robust, highly polymorphic, cost effective and have potential for automation. Characteristics of PCR–based Markers They are stable phenotypic ally, neutral and not influenced by the environment. Highly polymorphic, simple inheritance (often co– dominant), Easy and fast to detect as they are numerous and randomly distributed throughout the genome Minimum pleiotropic and epistatic effects Can be analysed at all developmental stages of the organism Requires small amounts of DNA, even pulverised or fossilised DNA can be used Their assay is more easily automated in handling, which increases both time and cost efficacies Early onset of phenotypic expression Non destructive assay Complete penetrance
It is extremely difficult to find a marker which fulfils all the aforementioned properties. Depending on the type of study to be undertaken a marker system can be chosen that would fulfil at least few of the above characteristics. Random Amplified Polymorphic DNA (RAPD) Random Amplified Polymorphic DNAs (Figure 1) are generated by applying the polymerase chain reaction to genomic DNA using random oligonucleotides as primers (Welsh and Mc Clleland, 1990). Due to short length, the primers have a possibility of annealing at a number of locations in the genome. Polymorphism arises due to changes in the primer-binding site in the DNA sequence (Sujatha et al., 2010). Isolation and purification of genomic DNA Annealing of decamer primers at two different sites on complementary strands of the DNA template Polymerase Chain Reaction (PCR) is allowed to run Amplified fragments are separated on agarose gel, stained with ethidium bromide and are visualised under a UV Transilluminator.
Variations in RAPD Technique DNA Amplification Fingerprinting (DAF) The DAF technique involves single arbitrary primers of less than ten bases, (Caetano-Anolles and Bassam, 1993) and the amplified fragments are visualised on polyacrylamide gel along with silver staining. The banding patterns obtained are reproducible. Arbitrary Primed PCR (AP-PCR) Developed by Welsh and McClelland (1991) it employs primers of 10–50 bases in length. Low stringency PCR amplification for the first two cycles of amplification in the initial two PCR cycles is carried out at non stringency conditions. Fragments amplified are visualised by autoradiography and are structurally similar to the RAPD products.
Advantages Requires small amount of DNA Non-radioactive assay No prior knowledge of the DNA sequence is required for primer designing Efficient in developing large number of DNA markers in a short time Simple, fast and relatively cheap
Disadvantages Microsatellites Microsatellites (Litt and Lutty, 1989) or STR (Short Tandem Repeat) or SSR (Simple Sequence Repeat) are 1– 6 bp long repetitive stretches of DNA present randomly in both Eukaryotes and Prokaryotes. They comprise di-, tri-or tetranucleotide repeats arranged in tandem arrays (Figure 2). The high polymorphic nature of SSRs corresponds to a higher mutation rate compared to other neutral regions of DNA which affects the number of repeat units. In eukaryotes (CA)n and (GA)n are the most abundant dinucleotide repeats (for e.g., in humans (CA)n repeat occurs once in every 30 kb). Although microsatellites are found in both non-coding and coding regions, their frequency is higher in transcribed regions. The variation in the number of repeats among different genotypes gives rise to lengthy polymorphism, which makes this marker suitable for determining genetic diversity, identifying important genetic traits, study of fine population structure, mating systems pedigrees, in forensics, and paternity studies. Variation in the number of tandemly repeated units arises due to strand slippage during DNA replication where the repeats allow matching through excision or addition of repeats (Schlotterer and Tautz, 1992). On either side of the microsatellite locus are the flanking regions, which are generally conserved (i.e., identical) across individuals of the same species and sometimes of different species (Selkoe and Toonon, 2006). A forward and reverse primer on either side of the microsatellite can be designed to amplify a fairly short (100–500 bp) region that can be easily detected on high resolution gels (for e.g., sequencing gels). The use of fluorescent labelled microsatellite primers and laser detection in genotyping procedures has significantly improved the throughput and automatisation (Wenz et al., 1998). Advantages They are simple, fast and highly polymorphic Evenly distributed throughout the genome Co-dominant and highly reproducible markers can be readily shared between different labs Little amount of DNA is required and also works on old or degraded DNA. Locus-specific (in contrast to multi-locus markers such as mini satellites or RAPDs) Highly variable as they provide a considerable pattern Many alleles can be identified at a single locus Analysis can be semi-automated and performed without the need of radioactivity
Disadvantages For most species, microsatellite markers must be developed de novo, which requires construction of a genomic library enriched for repeated motifs, prior characterisation of flanking region for primer design, optimisation of PCR amplification for each primer pair set. Most of these steps are either labour-intensive or time consuming. Inter-Simple Sequence Repeat (ISSR) It is a variant of PCR based on amplification of inter microsatellite sequences (Zietkiewicz et al., 1994). It employs oligonucleotide primers complementary to a simple sequence repeat region to amplify DNA segments present at an amplifiable distance in between two identical microsatellite repeat regions oriented in the opposite direction (Semagn et al., 2006). ISSR primers can be di-, tri-, tetra- or penta-nucleotide repeats. Limitless primers can be generated for various combinations of di-, tri- or tetranucleotides [(4)2=16, (4)3=64,] etc. ISSR primers are longer (14 or more base pair) than RAPD primers thereby lowering the risk of nonspecific primer annealing. The specificity of priming can be increased by the addition of anchor nucleotides at the 3’ or 5’ ends (Figure 3). Introduction of anchor ensures that the primer binds only to one end of a complementary SSR locus. This technique is fast, simple, highly polymorphic and more reproducible than RAPD. Variations in ISSR DAMD (Directed Amplification of Mini satellite region DNA) It employs a single primer which contains only the core motif of a mini satellite. SPAR (Single Primer Amplification Reaction) uses a single primer holding only the core motif of a microsatellite.
Advantages Microsatellite sequence-specific Does not require prior knowledge of the DNA sequence Robust, highly polymorphic and provides information about many loci simultaneously. Very useful for DNA profiling, especially for closely related species
Disadvantages Dominant markers and comigrating bands may not be homologous Polyacrylamide gel electrophoresis and detection with silver staining or radioisotopes may be needed Optimisation takes a long time
Aflp This is a DNA fingerprinting technique which is a combination of RFLP and PCR. AFLP technology was developed by Vo s et al., 1995. It is based on a selective amplification of a subset of restriction fragments obtained from a complex mixture of restricted digested genomic DNA fragments (Figure 4). The technique involves the following steps: Restricted digestion of genomic DNA with a 4 bp rare cutter (Eco R I) and a 6bp frequent cutter (MSe I) Ligation of double stranded oligonucleotide adaptor to both the ends of the restricted products Pre-amplification with primers complementary to the adaptors and having a single selective nucleotide Final (selective) amplification employing primers with up to three selection nucleotides Banding profiles are viewed on denaturing polyacrylamide gels either through auto radiography or fluorescence methodologies.
They originate by SNPs (Single Nucleotide Polymorphism) that create or remove restriction endonuclease recognition sites. The AFLP technique can generate 50–100 bands per assay which depends on the number of selective nucleotides in the AFLP primer combination, the selective nucleotide motif, GC content and genomic size. Advantages AFLPs are stable, have high genomic abundance and good reproducibility Large number of loci can be generated in a very short time Generates fingerprints of any DNA regardless of its source, and requires no prior knowledge of sequence data for primer construction
Disadvantages Dominant markers Requires purified and high molecular weight DNA Requires the laborious preparation of template DNA for each individual rather than being applicable to total genomic DNA It can be difficult to develop locus-specific markers from individual fragments Procedure is time consuming and requires the running of a DNA sequencing gel
APPLICATIONS OF PCR BASED MARKERS Genetic Fingerprinting Genetic fingerprinting is also known as DNA fingerprinting, DNA typing, and DNA profiling. It is based on the polymorphisms occurring at the molecular level, that is, on the base sequences of the genome. DNA variation is the substrate of genetic fingerprinting. DNA fingerprinting has come a long way from being just a forensic tool. It is useful in plant breeding. It has helped in identifying the plant genomics and markers for traits, identification of gene diversity, variation, etc. Many endangered plant species can be kept alive with the help of DNA fingerprinting. The advent of polymerase chain reaction (PCR) ushered a revolutionary approach in producing genetic fingerprints. The most popular or widely used techniques are RAPD, ISSR, SSR and AFLP. Random Amplified Polymorphic DNA was used for the genetic fingerprinting of 11 plant species, Andrachne telephioides, Zilla spinosa, Caylusea hexagyna, Achillea fragrantissima, Lycium shawii, Moricandia sinaica, Rumex vesicarius, Bassia eriophora, Zygophyllum propinquum subsp migahidii, Withania somnifera, and Sonchus oleraceus, collected from various areas of Saudi Arabia (Arif et al., 2012). A total of 164 bands were observed for 11 plant species using five primers. The highest pair-wise similarities (0.32) were observed between A. fragrantissima and L. shawii when five primers were combined. The lowest similarities (0) were observed between A. telephioides and Z. spinosa; Z. spinosa and B. eriophora; B. eriophora and Z. propinquum. They concluded RAPD as a reliable method to differentiate among all the plant species. Information thus procured can be further used for identification, conservation and sustainable use of these plants. Inter-Simple Sequence Repeat molecular fingerprinting markers have been employed to authenticate seven populations of Chimonanthus grammatus collected from China (Jiang et al., 2012). A high population differentiation (Gst = 0.249) was identified among these populations and significant association were found between genetic and geographical distances (r = 0.712, p < 0.001). Analysis of molecular variance (AMOVA) also revealed that 26.4% of the ISSR variation resided among populations while 73.6% resided within populations and 21.8% of the ISSR variation resided among Anyuan and Huichang groups while 78.2% resided within groups. ISSR proved as an effective and useful tool for understanding genetic diversity and population structure of C. grammatus which can provide an insight into the conservation and management of this species. SSR markers were used for genetic diversity analysis and DNA fingerprinting of 30 genetic stocks of tomato (Solanum lycopersicum L.) (Dhaliwal et al., 2011). Of the 25 primers used, 21 showed polymorphism and amplified 60 alleles. Similarity coefficient between any two genotypes estimated based on DNA amplification by SSR primers varied from 0.18–0.94. The lowest similarity coefficient (0.18) observed between genotypes belonging to the cultivated species lycopersicum and the wild species pimpinellifolium confirmed their differentiation at the species level. UPGMA revealed that SSR markers were helpful in differentiating the genotypes on the basis of horticultural and genetic factors. Based on the DNA fingerprints it was possible to differentiate 23 of the 30 genotypes screened. There is an existing demand in the herbal drug industry for an endorsement of the Zingiber sp in order to facilitate their commercial use as genuine phytoceuticals. Ghosh et al. (2011) used AFLP to produce DNA fingerprints for three Zingiber species. Sixteen collections, (Z. officinale, Z. montanum and Z. zerumbet) were used in the study. A total of 837 fragments were produced by seven primer pairs. Species-specific markers were identified for all three Zingiber species. The dendogram analysis generated from AFLP patterns showed that Z. montanum and Z. zerumbet are phylogenetically closer to each other than to Z. officinale. The AFLP fingerprints of the Zingiber species could be used to authenticate Zingiber sp-derived drugs and to resolve adulteration related problems faced by the commercial users of these herbs. Gene Tagging and Marker Assisted Selection Plant breeders make use of markers as a source of genetic information on crops as well as for indirect selection of traits to which the markers are linked. Some agriculturally important traits like yield, quality and some forms of disease resistance are controlled by many genes and are referred as quantitative traits. The regions within genomes that contain genes linked with a particular quantitative trait are known as quantitative trait loci (QTLs). In conventional breeding the markers were commonly the visible morphological characters and the breeders spend considerable time and effort in refining the crosses as the tight linkage or association of the desired characters with the apparent phenotypic characters was never clearly established. Therefore, identification of QTLs based only on conventional phenotypic assessment is not possible. Development of molecular markers created opportunities to select QTLs for the characterisation of quantitative traits. The use of molecular markers in plant breeding has given rise to “molecular breeding”. Molecular breeding comprise chiefly “gene tagging” followed by “marker-assisted selection” of desired genes or genomes. Gene tagging refers to the introduction of new DNA or the identification of existing DNA that can function as a tag or label for the gene of interest. Economic yield and adaptability can be improved by altering plant types. RAPD can be used for identifying and tagging plant type genes without much environmental interference. TT44-4 with open-tall and TDI2004-1 with compact-dwarf plant types were crossed to obtain F1 and a F2 population in pigeonpea. Dhanasekar et al. (2010) used this plant material and carried out an investigation for identifying and tagging RAPD markers linked with the plant type trait. RAPD analysis was performed followed by bulked segregant analysis (BSA). Two markers (OPF04700 and OPA091375) were identified that were present in the open-tall plants, which were absent in compact-dwarf plants. Linkage analysis revealed that these markers were located 8.4±0.03 cm and 9.6±0.032 cm respectively away from the plant type gene locus. RAPD proved very informative in identifying and tagging the compact-dwarf plant type trait. Inter-Simple Sequence Repeat polymorphism has been used for finding markers linked to clusters of disease resistance genes. Marczewski (2001) employed ISSR and detected markers linked to the Ns gene, responsible for a resistance of potato (Solanum tuberosum L.) to potato virus S (PVS). UBC811(660) and UBC811(950) were found to be linked to Ns. Linkage distances were estimated to be 2.6 and 6.6 cm respectively. These ISSR markers can be a powerful tool for detection of genotypes carrying the Ns gene in diploid potato breeding programmes. Molecular markers linked to wheat powdery mildew resistance gene Pm21 were identified by Qi et al. (2010). Preferred small groups (PSG) analysis was conducted using F2 segregating population cross between the wheat germplasm line CB033 with Pm21 and a common wheat variety Huixianhong. 102 markers showed polymorphism between the parents when screened with 258 SSR or STS markers. Two EST-SSR markers (Xcfe164 and Xedm129) and one STS marker (Xcinau188) showed good stability between the genotype and the phenotype to mildew in PSG. Linkage relationship between Pm21 and molecular markers was then established. Accuracy of the linkage between Pm21 and molecular markers were tested via F2:3 families and results showed that the three markers were reliable new markers linked to Pm21. The results obtained can facilitate molecular marker-assisted selection in breeding program. Fusarium oxysporum F.sp. melonis cause Fusarium wilt in melon. Wang et al. (2000) identified AFLP markers linked to resistance genes that can serve as a valuable tool for the selection of Fusarium wilt resistant genotypes. Fom-2 genes confer resistance to races 0 and 1 of the fungal pathogen. Homozygous resistant or homozygous susceptible progeny of F2 cross between MR-1 and AY was screened using 240 PstI/MseI and 200 EcoRI/MseI primer combinations to identify AFLP markers linked to Fom-2. Fifteen markers potentially linked to Fom-2 were identified all with EcoRI/MseI primer pairs. These were mapped relative to Fom-2 in a backcross (BC) population of 60 progeny derived from MR-1 × AY with AY as a recurrent parent. Two AFLP markers (ACT/CAT1 and AAC/CAT1) flanked the gene at 1.7 and 3.3 cm, respectively. These markers may therefore be useful in marker-assisted breeding programs. Somaclonal Variation and Genetic Instability Somaclonal variation is defined as genetic and phenotypic variation among clonally propagated plants of a single donor clone (reviewed in Sunderland, 1973; Duncan, 1997; Olhoft and Phillips, 1999). Somaclonal variation caused by the process of tissue culture is also called tissue culture-induced variation. Somatically stable variation includes phenotypes such as habituation of cultures and physiologically induced variation observed among primary regenerants. This type of variation is often not transmitted to subsequent generations. Meiotically heritable variations are important when the end product of the tissue culture is propagated and sold as seed. Mechanisms producing both, somatically and meiotically heritable variation also contributes to the decline in vigour and regenerability of cultures over time. The loss of culture health with time is a major detriment to the efficiency of transgenic plant production and much effort has been devoted to circumvent this problem. Study of somaclonal variation is relevant to applications such as in vitro plant propagation and plant transformation. In addition, somaclonal variation is likely a reflection of response to cellular stress. Therefore, understanding the mechanism of tissue culture variation will be useful in defining cellular mechanisms acting in the process of evolution and in elucidating the mechanism by which plants respond to stress. The roles of RAPD and SSR markers in detecting somaclonal variation of cotton (Gossypium hirsutum L.) were evaluated (Jin et al., 2008). 28 embryogenic cell lines via somatic embryogenesis and 67 regenerated plants from these embryogenic calli were selected and used for RAPD, SSR, chromosomal number counting and flow cytometric analysis. Both techniques were able to amplify all of the cell clones, and regenerated plantlets genomes and relative higher genetic variation could be detected in the culture type with 2, 4-D and kinetin hormone combination. The result suggested that 2, 4-D and kinetin hormone combination could induce relatively high somaclonal variation, it also suggested that RAPD and SSR markers are useful in detecting somaclonal variation of regenerated cotton plants through somatic embryogenesis. DNA variations of 48 Eucalyptus globulus plants regenerated by successive culture from seven different explants were assessed by AFLP analysis using 18 primer combinations (Mo et al., 2009). The proportion of polymorphic fragments did not correlate with the numbers of the regenerated plants. However, the more number of times the successive culture was done the more number of polymorphic bands was observed within the groups. AMOVA analysis showed 39.33% of the variation was found among the accessions that originated from different calli while 60.67% was from the same calli. The increasing utilisation of synthetic (encapsulated) seeds for germplasm conservation and propagation necessitates the assessment of genetic stability of conserved propagules following their plantlet conversion. Lata et al. (2011) assessed the genetic stability of synthetic seeds of Cannabis sativa L. during in vitro multiplication and storage for six months at different growth conditions using ISSR DNA fingerprinting. Of the 14 primers tested nine produced 40 distinct and reproducible bands. All the ISSR profiles from in vitro-stored plants were monomorphic and comparable to the mother plant, which confirms the genetic stability among the clones. GC analysis of six major cannabinoids showed homogeneity in the re-grown clones and the mother plant with insignificant differences in cannabinoids content, thereby confirming the stability of plants derived from synthetic seeds following six months storage. Phylogenetic Analysis Phylogenetic analysis through molecular methods has provided a powerful tool for the study of the evolutionary history of organisms, their genes and genomes. It involves construction of phylogenetic trees on the basis of molecular data. This information can be further employed in understanding systematics and evolution of green plants. Random Amplified Polymorphic DNA and ISSR markers were employed as genetic markers for the assay of the genetic relationship of four Ficus cultivars namely, benjamina, hawaii, stipulata and nitida (Hadia et al., 2008). According to the RAPD analysis, 10 primers gave a total of 340 amplified fragments, in which 212 (62.4%) were polymorphic. Out of 340 RAPD–PCR fragments, 62 were found to be useful as cultivar specific markers. In ISSR analysis, 11 of the tested ISSR primers generated 179 polymorphic bands out of 299 fragments. 50 DNA amplified fragments were considered as cultivar-specific markers. Results of the combined data exhibited that the two most closely related cultivars were FH and FS with the highest similarity index (0.618) while the two most distantly related cultivars were FS and FN with low similarity index (0.387). It was concluded that RAPD and ISSR polymorphisms could be used as efficient tools for the detection of similarities and phylogenetic relationships of the genotypes, which could be useful in the breeding programs. Phylogenetic relationships among interfertile species of Trollius L. (Ranunculaceae) were investigated using nuclear and chloroplast DNA sequences and AFLP markers (Despres et al., 2003). ITS sequences were not informative at the intrageneric level, but confirmed the sister relationship between Trollius and Adonis genera. It provided new information on the phylogenetic relationships among five Ranunculaceae genera where as chloroplast DNA was more informative among Trollius species, but not consistent with the sections described previously. AFLP proved to be a powerful tool to resolve the complex genetic relationships between the morphological entities constituting the genus Trollius. SSR markers were used to evaluate phylogenetic relationships among 14 genotypes of Actinidia species (Korkovelos et al., 2008). The genetic similarity of the genotypes calculated with Jaccard and/or Dice similarity coefficients varied from 0.100–0.579, indicating a broad genetic base for the genetic material tested. Eight species were clustered in two main groups and each main group in two subgroups. Data showed a close genetic relationship among Actinidia chinensis and Actinidia deliciosa species. Data showed that the di-nucleotide microsatellites were more polymorphic, than the tri-nucleotide and penta-nucleotide, and were more efficient in establishing genetic similarities. Assessment of Hybridisation Hybridisation is considered to be wide spread among plants and is a prominent factor in evolution which may trigger the differentiation of new lineages. The introgression, the movement of genes from one species into the gene pool of another by backcrossing between the interspecific hybrid and one of its parents, is one of the principal evolutionary consequences of reproduction in the wild plants. Hybrids usually grow intermingled with their original species. In polymorphic populations, introgression may go unnoticed because appropriate methods for its detection are not used or few diagnostic features separate parental taxa. Morphology, comparative anatomy and physiology have been previously employed for identifying plant hybrids, however, now a days confirmation of a hybrid origin of a species is increasingly been done by molecular techniques. The investigation pertaining to the hybrid identification in Gossypium hirsutum L. through RAPD analysis was conducted by Ali et al. (2008) on three genotypes (CIM-511, SLS1 and Paymaster) and their hybrids (SLS1 × CIM-511, Paymaster × CIM-511, Paymaster × SLS1). 16 primers amplified a total of 518 fragments in the parents and hybrids out of which 76 loci were polymorphic. Comparison of the RAPD banding pattern of the parents with the respective hybrids clearly identified genuine hybrids. This study suggested that RAPD analysis can be utilised for both reliable and less time consuming identification of hybrids. Crossbreeding is an efficient means to increase production and quality in plants. Lin et al. (2010) crossbred two bamboo species Phyllostachys kwangsiensis (female parent) and Phyllostachys bambusoides (male parent) and obtained bamboo hybrids. Two bamboo hybrids were identified using PCR/ISSR. ISSR markers were proved useful to identify bamboo hybrids. Hybrid identification of 16 sunflower hybrids was confirmed using simple-sequence repeat methodology (Iqbal et al., 2010). Of the 20 specific SSR markers, 18 authenticated the purity of hybrids; the remaining two specific primer pairs gave ambiguous DNA fragments. The results indicated that SSR analysis for the identification of hybrids derived from the crossing of different inbred sunflower lines could improve the accuracy of selection, save time and reduce cost. Using 174 amplified fragment length polymorphism (AFLP) markers Lubell et al. (2008) distinguished between 43 Berberis thunbergii cultivars and analysed the genetic similarity of 62 B. thunbergii genotypes, B. julianae, B. koreana, B. vulgaris and B. vulgaris ‘Atropurpurea’. Seven different accessions of B. thunbergii ‘Crimson Pygmy’ from a variety showed identical AFLP profiles, indicating that there was only one genotype in cultivation and not a collection of similar genotypes. A dichotomous identification key was developed for 42 B. thunbergii cultivars, hybrids and B. vulgaris ‘Atropurpurea’ using 23 markers. Thus cultivars can be identified that are difficult to distinguish visually, and to test whether prospective cultivars are unique genotypes or sub-clones of established cultivars. RAPD, ISSR and CCMP analyses revealed that ochroleuca and mexicana shared many similar traits but the molecular data suggests that these two species have evolved independently. This fact is also supported by their diverse geographical distribution. Mexicana is introduced into all tropical and subtropical regions of the world; ochroleuca is introduced into Australia and India recently. Karnawat and Malik (2011) carried out investigations to assess genetic variation in A. mexicana, A. ochroleuca and natural ‘hybrids’. The RAPD primers (100) tested revealed 57.89% polymorphism with 121 polymorphic bands out of 209 between the species. Dendrograms based on UPGMA and PCO analysis clearly indicated the hybridity of natural plants (triploid) bearing light lemon color flowers. A close relationship between mexicana and hybrid is indicated. ISSR primers made interesting revelations and supported the hybrid nature of the natural triploid. A close scrutiny of dendrograms, based on UPGMA and PCO analysis, indicated the hybrid nature of the natural plant bearing LCF. Genetic closeness of the hybrids to A. ochroleuca also emerged. Two dimensional scaling of the three argemones by principal coordinate analysis using the Jaccard's coefficient based on the pooled data of RAPD and ISSR primers showed close clustering between ochroleuca and the two hybrids. Comparative analyses involving RAPD and ISSR are very pivotal; the former is efficient compared with ISSR assay with regards to detection of polymorphisms since RAPD can detect nearly 85% as compared with 76% of ISSR markers. Moreover, mean number of polymorphic bands per primer and total number of polymorphic bands are more for RAPD than ISSR. There are several parameters for example average heterozygosity, gene flow estimates, which are more in ISSR than RAPD markers. It is of special interest to mark that RAPD data is more close to ISSR and RAPD combined data, as it would be interesting to evaluate diversity with RAPD and ISSR. When the comparison of RAPD and ISSR derived dendrograms was made, it was observed that the pattern of clustering of the genotype remained nearly the same in ISSR and combined data of RAPD and ISSR. Their values were also significantly different from each other. Thus, both ISSR and RAPD markers are required to be used separately as efficient marker systems in Argemone taxa because of their potential to reveal several bands in a single amplification. The PCO analysis and dendrogram based on UPGMA exhibited close genetic variation in the latter. The genetic closeness points towards the role of ochroleuca in the origin of the hybrid. Narrow genetic base of Argemone sp. could probably be due to the autogamy and genetic diversity, which will be understandably low and will reflect on the mode of propagation and reproductive behaviour. The ten ccmp primers, with the exception of ccmp 8 and ccmp 9, yielded a single discrete PCR product. Likewise, ccmp 8 failed to produce amplification products with templates from Solanaceae, Actinidia, Cruciferae and monocotyledonous sp. The ccmp primers were able to distinguish all the Argemone taxa with a unique set of alleles and also aided in unraveling the direction of the interspecific crosses using OPQ-5, OPQ-9 and OPQ-12. It was possible to mark out specific molecular profiles associated with maternal and paternal parents. This was further confirmed through Consensus chloroplast microsatellite primers (CCMP 1,2,3,4,5,6,7,8,9,10). It was possible to generate allele sizes of amplification products from Argemone species. With the exception of ccmp 1 and 3 all other primers gave amplification product, which was different from that of tobacco. The ccmp 3 primer confirmed that A. mexicana is the paternal and A. ochroleuca as the maternal parent. Allele length of the two hybrids, AH and BH, with ccmp 3 primer was the same as that of the maternal parent thus confirming A. ochroleuca as the maternal parent. Plants being lemon coloured flowers were subjected to RAPD, ISSR, ccmp analyses and were diagnosed as triploids where ochroleuca was a maternal and mexicana was a paternal parent. To the best of our knowledge no such study on genetic diversity, using molecular markers is available in the genus Argemone. Present investigations have attempted to examine the level of genetic variation within mexicana-ochroleuca complex. The two marker systems have been employed to evaluate genetic diversity and to seek deep insight on the phylogenetic relationship of the two species and the triploid natural hybrid. Genetic Mapping Heritability is greatly affected by the genetic architecture of the desired trait, which is described by the number of genes, the degree of their effects, and the type of gene function linked with phenotypes. Comprehension of genetic architecture and gene function often has the greatest impact on achieving genetic gain. Linkage analysis reveals the arrangement and distribution of molecular markers on genome. Markers that co-segregate must be linked, that is they must be located in each others vicinity on the genome. Such analysis can finally result in the construction of a genetic map, on which all markers are arranged in separate linkage groups or chromosomes. The extent of linkage reflects distances between markers. Mapping studies provide information on number of loci, allelic effects and gene action controlling desired traits. More importantly quantitative trait loci (QTLs), that is, genomic segments that contain quantitative traits can be identified. Towards a better understanding of the genetic basis of kenaf, for the improvement of production and to lay the foundation for molecular breeding efforts, a primary genetic linkage map was constructed using sequence-related amplified polymorphism (SRAP), ISSR and RAPD (Chen et al., 2011). ‘Alian kenaf’ and ‘Fuhong 992’ were used as parents to construct an F2 population consisting of 180 plants. 494 SRAP, 60 ISSR, 120 RAPD and 300 two-primer RAPD mixture primers amplified 396 polymorphic loci in total. These 396 loci were used to construct the genetic linkage map, a total of 307 loci were grouped into 26 linkage groups. These markers were distributed randomly in all linkage groups without any clustering. The genetic map constructed using SRAP, ISSR and RAPD could be utilised for further genetic studies. A high-density genetic linkage map in cotton was constructed using large number of EST-SSRs (Yu et al., 2011). The map comprised 2316 loci on the 26 cotton chromosomes, 4418.9 cm in total length and 1.91 cm in average distance between adjacent markers. Such SSR developed maps are of great value for comparative genetics and to accelerate marker-assisted selection (MAS). Genetic linkage maps of European larch (Larix decidua Mill.) and Japanese Larch larix kaempferi (Lamb.) Carr. was constructed using segregation data from 112 progeny individuals of an hybrid family (Arcade et al., 2000). A total of 266 markers (114 AFLP, 149 RAPD and 3 ISSR loci) showing a testcross configuration, that is heterozygous in one parent and null in the other parent, were grouped. The maternal parent map (L. decidua) consisted of 117 markers separated within 17 linkage groups (1152 cm) and the paternal parent map (L. Kaempferi) had 125 markers gathered into 21 linkage groups (1206 cm). The PCR-based molecular markers facilitated the construction of genetic maps, thus ensuring a good coverage of the larch genome for further QTL detection and mapping studies. Molecular Pharmacognosy Molecular pharmacognosy combines pharmacognosy and molecular biology. It provides an independent approach for the characterisation of medicinal plant materials at the DNA level. The main objective of this type of research is to achieve the identification and quality assessment of individual plants and plant populations, or provide scientific bases for the production and sustainable use of medicinal raw material resources. PCR based markers have been widely used for authentication of plant species of medicinal importance. This is especially useful in case of those that are frequently substituted or adulterated with other species or varieties that are morphologically and/or phytochemically indistinguishable. It is important for the development of better quality herbal drugs, thus providing quality control. Random Amplified Polymorphic DNA technique was employed for authentication of Piper nigrum (black pepper) from its adulterant Carica papaya (Khan et al., 2010). Out of eight primers used, five gave species-specific unique amplicons, which could clearly discriminate genuine as well as adulterant samples having similar morphology. RAPD could thus, help to serve as a complementary tool for quality control of genuine samples sold in the local markets. Inter-Simple Sequence Repeat molecular fingerprinting markers have been employed to authenticate three genuine species of rhubarb, Rheum officinale Baill., Rheum palmatum L., and Rheum tanguticum Maxim, using 15 primers (Wang, 2010). A total of 155 DNA fragments were amplified, of which 132 were polymorphic (85.2% of all bands). Four specific authentication markers were detected to authenticate three species of rhubarb. Three loci combinations from primer UBC816 with any one of the primer UBC807and UBC811, that is UBC816-620bp with UBC807-520bp, UBC816-620bp with UBC807-400bp, and UBC816-620bp with UBC811-248bp served the purpose of distinguishing three genuine species of rhubarb. Swertia chirayita is often adulterated with other less potent Swertia spp. Misra et al. (2010) used AFLP markers to produce DNA fingerprints for six Swertia species. 19 accessions were used, which engaged 46 useful AFLP-selective primer pairs that generated 5312 fragments. Species-specific markers were identified for all six Swertia species. These AFLP fingerprints of the Swertia species could be used to authenticate drugs made with these species and to resolve adulteration-related problems faced by the commercial users of these herbs. Genotoxic Effects of Heavy Metals Accumulation of heavy metals in the environment is a serious global concern. It influences physiological systems of plants and other living organisms. PCR based markers have been used as bio indicators and biomarkers in detection of genotoxic effects of heavy metals. Random Amplified Polymorphic DNA fingerprinting technique was used to detect DNA damage in the kidney-bean seedlings treated with two selected heavy metals at concentrations of 150 and 350 mg x l(−1) (Enan, 2006). Polymorphism was observed when treated samples were compared with the untreated ones. Results suggested that a qualitative measure reflecting changes in RAPD profiles were significantly affected at higher concentrations of the tested heavy metals. Comparison between untreated and treated genomes revealed that RAPD analysis can be used to evaluate how the environmental pollutants modify the structure of DNA in living organisms. Three heavy metals Zn, Pb and Cd showed a dose-dependent effect on radicle and coleoptile lengths of E. sativa (Qurainy, 2010). The ranking of genotoxic potencies in all three heavy metals was in the descending order: Cd2+ > Pb2+ > Zn2+. Twenty ISSR primers were used, of which four did not amplify; three gave a single band and the rest of the thirteen primers generated up to six bands. Sixteen primers exhibiting amplified products gave monomorphic bands; only two primers (OPC-5 and OPC-7) gave unique extra bands in seedlings treated with medium and high concentrations of heavy metals Cd and Pb, respectively. The dendrogram was constructed to evaluate the genetic distance generated among the seedling treated with various heavy metals at various concentrations. The similarity matrix values were found from 42.8%–100% and these values showed the genetic divergence among the seedlings treated with various concentrations of heavy metals. Amplified fragment length polymorphism (AFLP) and selective amplification of polymorphic loci (SAMPL) tests revealed dose-related increases in sequence alterations in plantlets exposed to 10–200 mg/l potassium dichromate (Labra et al., 2003). Individual plantlets exposed to chromium under similar conditions showed different AFLP and SAMPL DNA profiles. MSAP analysis showed extensive methylation changes in CCGG-sequences, with the net result being genome-wide hypermethylation. These results showed a clear DNA alteration in plants as a response to chromium exposure and the effect was dose-dependent. DNA polymorphism detected by different markers supports the effectiveness of the use of PCR based tools for the investigation of environmental toxicology and for evaluating the concentration of pollutants by DNA analysis in plants.
Top Figures | Figure 1:: Steps involved in Random Amplified Polymorphic DNA (RAPD)
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| | Figure 2:: Generation of SSR Length Polymorphism (i) PCR amplification of an SSR locus: (GA)n in three diploid genotypes: AA, BB and AB. (ii) Gel pattern of the amplification products with different combination of alleles
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| | Figure 3:: Represents ISSR-PCR with a 3’anchored primer and 5’anchored primer
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| | Figure 4:: Shows steps involved in AFLP
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