Page | 123
16
Page | 124 people, are widely reported in the media and are of interest to the general population. DNA fingerprinting technology has now been extended from humans to even plants. In plants, identification of cultivars is one area where DNA fingerprinting is being used routinely and has applications in protection of plant breeders’ and farmers rights.
Cultivar fingerprinting
The question underlying the use of DNA fingerprinting data is what is the probability that two or more cultivars have the same genetic fingerprint/ profile?
If we could sequence and compare the DNA of each cultivar, we would find that all cultivars are different. Even natural clones, such as identical twins in humans, will have acquired a few differences that are mutations in their DNA, during their growth from a single cell to an adult.
When it is not possible to sequence the entire individual's genome then instead, we rely on differences in length of short stretches of DNA at different loci commonly called markers.
These stretches of DNA can be visualized as shown in the figure. Based on this figure can we say that any two cultivars are identical? Can we assign a numerical value to our answer? Our answer to this question depends on the probability of two or more cultivars having the same pattern of these DNA fragments, the same sized SSR markers. The SSRs chosen for DNA fingerprinting purposes are inherited independently of each other. They are on different chromosomes. Therefore, in theory, if the frequency of SSR1 in a population is 1 in 50, and SSR2 has a frequency of 1 in 1000, then only 1 in 50,000 cultivars would be expected to have both these SSRs. More SSRs (not two as in the present case) are usually monitored in each
"DNA fingerprint" and are chosen so that the probability of a false positive match is extremely low (Figure 1).
Figure 1: DNA profile, with SSR markers, of six cultivars with two primers
Page | 125 Choosing an appropriate methodology
There are different methodologies available for DNA fingerprinting such as RFLP, RAPD, ISSR, AFLP, SSR, SNP etc. The important criteria for choosing a methodology depends on the reproducibility of the data between laboratories, accessibility of detection platforms, repeatability over time, discrimination power and ease in creating database.
Database generation
As advancements in methodologies and new equipment are routinely happening, the continued sustainability of database should be an important consideration. This is particularly true for DNA sequencing data. Also, earlier radioactively labeled primers and sequencing gels were used to produce DNA fingerprinting data which now can be done using fluorescent labeling followed by separation on high throughput detection systems. Repeatability and reproducibility are important in the construction, operation and longevity of databases and is very important in generating a centrally maintained database, populated with verified data from different sources.
Mapped markers
With the availability of robust techniques, the trend has been to use mapped SSRs (microsatellites) and may be sequencing (Single Nucleotide Polymorphisms, SNPs) in near future. Other genic or functional marker techniques which rely on DNA sequence information may also fulfill the above criteria but their use in DNA profiling of plant varieties needs to be explored. A marker or set of markers selected for DNA profiling should be polymorphic and have repeatability within and reproducibility between, laboratories in terms of scoring data. To help avoid markers that may be linked, it is desirable to know the map position of the markers. It is also advisable to avoid markers with “null” alleles.
Microsatellite markers
Microsatellite or Simple Sequence Repeats (SSRs) make use of polymerase chain reaction (PCR) and has several advantages. SSR markers are expressed co-dominantly, are generally robust, repeatable, easy to score (record), and easy to automate for high-throughput detection.
Moreover, several SSRs can be multiplexed and detected by non-radioactive DNA sequences or on gel electrophoresis or capillary electrophoresis. With recent advancements in genomics, mapped SSRs are now available in several crops. For effective microsatellite analysis, selecting high quality SSR markers that are unlinked, polymorphic, reliable and reproducible and having no stuttering is essential. Primers for any kind of marker system should be synthesized by an assured supplier. Some SSRs are publicly available.
Plant material
The source and type of the material and how many samples need to be analyzed are the main issues with regard to the material to be analyzed. The plant material to be analyzed should be
Page | 126 an authentic, representative sample of the variety and, where possible, should be obtained from the sample of the variety used for examination for the purposes of Plant Breeders’
Rights or for official registration. Use of samples of material submitted for examination for the purposes of Plant Breeders’ Rights or for official registration will require the permission of the relevant authority, breeder and/or maintainer, as appropriate. The plant material from which the samples are taken should be traceable in case some of the samples subsequently prove not to be representative of the variety.
Sampling strategies
The type of plant material to be sampled and the procedure for sampling the material for DNA extraction will, to a large extent, depend on the crop or plant species concerned. For example, in seed-propagated varieties, seed or seedlings may be used as the source of DNA, whereas, in vegetative propagated varieties, the DNA may be extracted from leaf material.
Whatever the source of material, the method for sampling and DNA extraction should be standardized and documented. Furthermore, it should be verified that the sampling and extraction methods produce consistent results by DNA analysis. It is essential that the samples taken for analysis are representative of the variety. With regard to being representative of the variety, consideration should be given to the mode of propagation i.e.
whether self-pollinated or cross-pollinated. The size of the sample should be determined taking into account suitable statistical procedures.
Reference set
A DNA reference sample set of varieties can be created for use in different detection equipment in different laboratories and for appropriate size calling as molecular size standards behave differently in the various detection systems. This DNA reference sample set can be stored and also supplied to other laboratories.
References
Jeffreys AJ and V Wilson (1985) Individual-specific ‘fingerprints’ of human DNA. Nature 316 (6023): 76-79.
Larson S (2002) Plant Genotyping: The DNA fingerprinting of plants. Heredity 88: 220-220.
Nybom H, K Weising and B Rotter (2014) DNA fingerprinting in botany: past, present, future. Investig Genet. 5: 1 (doi:10.1186/2041-2223-5-1)
Rana MK and KV Bhat (2017) DNA Fingerprinting in Plants: Standard Operating Methods and Protocols. ICAR-National Bureau of Plant Genetic Resources, New Delhi, India, 73p (http://www.nbpgr.ernet.in/Divisions_and_Units/Downloadfile.aspx?EntryId=7432)
Weising K, H Nybom, K Wolff and G Kahl (2005) DNA Fingerprinting in Plants: Principles, Methods and Applications (2nd edn.), CRC Press, 472p.
Page | 127