Plant DNA Fingerprinting

Introduction to DNA Fingerprinting

Table 1: A Comparative Analysis of DNA Fingerprinting Techniques

Marker	Description	Advantages	Disadvantages
RFLP	In this method, unequal lengths of DNA fragments are produced by cutting VNTRs sequences up to 30 sequences long with restriction enzymes at specific sites. There are different VNTRs, as there are different plant species, number and location of restriction enzyme-recognition sites. The routine Southern blot experiment can be used. The hybridization results can be visualized by autoradiography or using chemiluminesence. RFLP markers are most suited to studies at the intraspecific level or among closely related taxa. In plant genetic analysis, RFLP markers were initially used for estimating genetic distance and fingerprinting in wheat and detection of genetic variability in rice. In plant and animal breeding, RFLP has been used in genetic dissection of maturity, identification of chromosomal regions responsible for regeneration of RFLPs, comparative mapping of chromosomes in wheat, rye and barley, analysis of wide compatibility in rice varieties, QTL mapping, gene tagging, and identification of ecotypes. RFLPs are also used to settle cases where biological parentage is at issue. Unlimited number of loci Co-dominant Many detection systems Can be converted to SCARs Robust in usage Good use of probes from other species Detects in related genomes No sequence information required Labour intensive Fairly expensive Large quantity of pure DNA needed Low levels of polymorphism Time consuming Needs considerable degree of skill
RAPD	This method is most commonly used for primary assay and useful for efficient screening of nucleotide sequence polymorphism between two species of plants using short single primer of arbitrary nucleotide sequence at low annealing temperatures during PCR. Amplified fragments are separated by gel-electrophoresis and polymorphisms are detected as the presence or absence of bands of particular size. The targeted band is identified and the DNA on agarose is isolated and sequenced. RAPD markers are used for characterization, estimation of genetic relatedness and determination of genetic diversity of tea germplasm and Ficus caricaL. clones. It is efficient tools for identification of markers linked to agronomically important traits. Results obtained quickly Cost effective than RFLP No sequence information required Relatively small DNA quantities required High genomic abundance Good polymorphism Can be automated Lack of specificity Highly sensitive to laboratory changes Low reproducibility within and between laboratories Cannot be used across populations nor across species
SSR	Simple sequence repeats or microsatellites are a class of repetitive DNA elements. It is very short (2-10 bp), middle repetitive, tandemly arranged, highly variable (hyper variable) DNA sequences dispersed throughout fungal, animal, human genome and abundant in plants, occurring on average every 6-7 kb. SSR is isolated by using hybridized probes and detected by gel electrophoresis using specific dyes or radiolabelling. SSR markers are used to reveal the large number of polymorphisms, with one locus in soybean (Glycine max) reported to have 26 alleles. It is also co-dominance markers which will increase the efficiency and accuracy of population genetic measures compared to AFLPs and RAPDs Fast Robust, efficient than RAPD Highly polymorphic Can be automated Only very small DNA required than RFLP High developmental and startup costs Species-specific Sometimes difficult interpretation because of stuttering Usually single loci even in polyploids
AFLP	This method is a PCR based derivative of RFLP. The sequences are selectively amplified using the P³³r non radioactive labeled primers. The mixture of amplified fragments are separated according to size in a polyacrylamide gel and visualized by means of autoradiography or other procedures for development of non radioactive systems. AFLP markers help in assessing genetic diversity among cultivars such as wheat, Triticum aestivum. It also helps detect higher level of polymorphism than RAPD or RFLP.	Small DNA quantities required No sequence information required Can be automated Can helps in determining a large number of polymorphism Cost effective Evaluation of up to 100 loci Marker clustering Dominant Technique is patented Can be technically challenging Require high quality of DNA sample

Adapted from Madhavan et al. (nd) and Vasudevan (2011).

The advantages of DNA fingerprinting in plants are as follows:

i.Protection of the ecosystem

DNA fingerprinting can be used as an effective tool for molecular breeding for commercially important (Figure 5) or endangered plant species and conserving the eco-diversity of our planet (Vasudevan 2011). Several international plant resource germplasm (genetic material) collection centers are exploiting DNA fingerprinting to help them focus their limited resources on maintaining and propagating those unique collections (Hong 2011).

Fig. 5: Two fast growing indigenous tree species, Kelampayan (left) and Sawih (right) polymorphic genetic markers have been developed by research group at the Forest Genomics & Informatics Laboratory (fGiL), UNIMAS. These markers could be then used for more accurate means of tree selection for plantation and improvement activities of the species.

ii.Identifying marker traits and genetic mapping

Some of the desirable traits that breeders try to incorporate into crop plants including better yield and quality, tolerance to environmental pressure (salinity, temperature, drought), microbe resistance and herbicide tolerance are more important to produce than others. DNA fingerprinting has been used as useful, rapid and affordable tool for selective breeding by identifying marker traits of interest. Since DNA fingerprints are taken from the same DNA that carries the entire genetic blueprint for the plant, pieces of DNA that are close together tend to be passed on together from one generation to the next. If one particular band of a DNA fingerprint is found to be inherited along with a useful trait (for example as shown in Figure 6), that band serves as a marker for that trait. This process is sometimes referred to marker-assisted selection (MAS). In MAS, it is easier and less expensive to select the plants harbouring the piece of DNA associated with a desirable trait rather than to grow them to maturity and see if they develop the desirable trait (Betsch 2004; Hagedorn nd).

		Parent 1	Parent 2	Offspring 1	Offspring 2	Offspring 3
Phenotype		Salt intolerant	Salt tolerant	Salt intolerant	Salt tolerant	Salt intolerant
Size (kb) 15 kb 10 kb 5kb	Molecular Ladder

Fig. 6: Parent 2 which is salt tolerant has same DNA fingerprint with offspring 2. Both of the plants show two bands, 15kb and ~13kb in size, respectively. Since the ~13kb band is not detected in salt intolerant parent fingerprint, the ~13kb band can serve as molecular marker for salt tolerant trait. In the case above, offspring 2 will be selected for large scale cultivation as it is salt tolerant.

Furthermore, molecular marker may help scientists determine the location (map) of genes that control important traits at regular intervals along the DNA chromosomes, like the mile-markers along interstate highways (Figure 7). After markers are located, virtually any observable characteristic could be traced to a specific stretch of DNA by routine laboratory procedures (Betsch 2004).

Fig. 7: Genetic markers (Betsch 2004)

iii.Identification of gene diversity, variation and mutations

DNA fingerprinting has proved accurately in genotypic characterization of plant species and strains. It is useful as most plants though belonging to the same genus and species, may show considerable variation between strains. A good example of this is the fraudulent adulteration of Chianti wines with inferior quality grapes (Vasudevan 2011). This is also the case with medicinal plants, where the amounts of active chemicals may vary from plant to plant. For example in turmeric planted in Malaysia, different variety shows different percentage of curcumin (bioactive principle) content (Table 2). There are also several cases reported that similar plants from different region show different level of bioactive compound correspond to the therapeutic effect of these plants. In such cases, there are observed variations in the genetic composition of the plant, in addition to varying amounts of the active drug compound. For example, the application of DNA fingerprinting in the study of quinine from Cinchona bark revealed the presence of this compound in the bark of Cinchona grown in the plains but no active quinine was found in the same species of tree grown on hilltops and slopes which looks morphologically similar (Vasudevan 2011).

Table 2: Turmeric variety planted in Malaysia

Adapted from Jabatan Pertanian Malaysia

In addition, DNA fingerprinting can serve as a tool in detection of mutated or genetically modified organism in agriculture based on current situation where new international rules are requiring crops which are genetically altered to be separated from ordinary crops (Hagedorn nd).

The genetic modified (GM) rice and maize can be detected using DNA fingerprinting method.

iv.Authentication and quality control of medical herbs and herbal drugs

The ability to identify seed varieties is important to guaranteeing the authenticity of a crop being purchased (Hagedorn, nd). A survey by a group of researchers at Temasek Life Sciences Laboratory on several herbs sold in Singapore found that some herbs of different genetic identities were sold under the same generic names. These findings show that reliable authentication and quality control for herbal materials are becoming critical for the protection of consumers, for the sustainable development of the industry, and for the integration of folk medicine into mainstream medicine (Hong 2011).

v.Protection of property rights for new plant varieties

Intellectual property rights (IPR) are defined as the rights granted by a state authority for certain products of intellectual effort and ingenuity. The different forms of IPR include patents, plant breeders’ rights, trade secrets, trademarks, copy rights, designs, know-how, geographical indication, material transfer agreement and farmers’ or community rights (Bhat nd). With the absence of property rights for new varieties, breeders derive little benefit since new plant varieties can be easily multiplied by seeds or vegetative propagation. With the ability of getting highly specific, precise and reliable DNA profile for a single plant in the short time, DNA fingerprint can prove that new varieties (developed by genetic engineering, tissue culture, or traditional methods) satisfy necessary criteria for granting protection. These criteria may include novelty, distinctiveness, uniformity, and stability. For administrators of plant property rights, DNA fingerprinting can help select most suitable reference varieties for morphological comparison and save cost. It is most effective in enforcing protection by proving infringement of property rights (Hong 2011).

Reference

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3. Bhat, K.V. nd. DNA Fingerprinting and Cultivar Identification. National Research Center on DNA Fingerprinting. Accessed on 2 Dec. 2011

4. Freeman, S. nd. How DNA Profiling Works in HowStuffWorks? Available at http://science.howstuffworks.com/dna-profiling1.htm Accessed on 2 Dec. 2011

5. Hagedorn, C. nd. Fields of Fingerprints: DNA Testing for Crops in Virginia Cooperative Extension. Available at http://filebox.vt.edu/cals/cses/chagedor/fingerprint.html Accessed on 1 Dec. 2011

6.Hong, Y. 2011. DNA Fingerprinting of Botanicals. Innovation. Vol. 10 (2)

7. Jabatan Pertanian Malaysia. nd. Panduan Menanam Kunyit. Available at http://www.pertanianperak.gov.my/jpp/images/pdf/kunyit.pdf Accessed on 2 Dec 2011

8. Jeanty, J. nd. How Does DNA Fingerprinting Help Plants? in eHow. Available at: http://www.ehow.com/about_6321793_dna-fingerprinting-plants.html Accessed on 2 Dec. 2011

9. Madhavan, V. T.P., Nair, A.M. & Hari.V.G. nd. DNA Fingerprinting in Plants in Biosolutions. Available at http://biosolutions.6te.net/articles_files/dna_fin.html Accessed on 2 Dec. 2011

10. Vasudevan, H. 2011. DNA fingerprinting in the standardization of herbs and nutraceuticals. The science creative quarterly. Issue 6. Available at http://www.scq.ubc.ca/dna-fingerprinting-in-the-standardization-of-herbs-and-nutraceuticals/ Accessed on 2 Dec. 2011