Defining
DNA marker technology
Dr
Syamal Ghosh, along with Dr CK Guha Sarkar, Dr M Shyamsunder Singh
and Dr NG Ramchandra, explain the intricacies of DNA marker technology
used extensively for cultivar identification
The
field of forensic science was revolutionised by the ability to extract
and type DNA from biological fluids left at a crime scene using molecular
techniques that identify individual DNA samples. These same techniques
can be applied to plant and seed identification. Identification of
plant varieties is essential in agriculture as WTO agreement envisages
protection of plant varieties by ‘sui generis’ system, such as Plant
Breeders Right (PBR) or Plant Variety Protection (PVP) Act provided
under the International Conventions for the Protection of new Varieties
of Plant (UPOV). According to PVP the new varieties must be distinct,
uniform and stable (DUS). The legal right to market a newly bred
cultivar depends on the results of statutory testing, which provides
information regarding its novelty, distinctness, uniformity and stability.
DUS testing guarantees the quality of the new cultivar for farmers
and merchants and describes that the new cultivar is distinct from
other released cultivars, uniform, stable as well as assures that
it is of correct genotype as specified by the breeder.
Moreover,
it is also used to protect PVP and encourages the continuous development
of new varieties. PVP have evolved since the introduction of the concept
of essentially derived varieties by UPOV in 1991. This has increased
since the development of genetic engineering which made possible the
introduction of a desired single gene into a variety.
Traditionally,
morphological markers have been used to identify varietal genotype
and genetic purity based on the assessment of a range of phenotypic
characteristics. Morphological markers may be very effective for many
purposes but have their own limitations. Morphological characters
used for cultivar identification include plant growth period, seed
shape among others. Major limitations on the use of morphological
characters includes subjectivity in the analysis of character, environmental
influences, limited diversity among cultivars, and some diagnostic
characters are limited to a particular stage of development, such
as flowering or fruit ripening. These stages usually require growing
plants to full maturity prior to classification and characterisation.
The
performances in VCU (Value for Cultivation and Use) trials such as
yields and flowering are not limited value and cannot be used for
cultivar identification. The difficulties are still more when large
number of cultivars with short commercial life span have to be evaluated
for discrimination. These considerations have led to the exploration
of other techniques including cytogenetic analysis, biochemical analysis
and DNA profiling techniques.
Limitations
of cytogenetical analysis
Cytogenetic
analysis has not been widely used and affords the necessary resolving
power in only a few species such as wheat, where the chromosomes have
been thoroughly analysed. According to International Seed Testing
Association (ISTA) the common rapid tests done by the laboratories
were biochemical and electrophoratic analysis. The biochemical method,
such as isozymes have been widely used for routine testing of parentage,
monitoring genetic purity and as descriptors in DUS along with morphological
characters. Electrophoresis of seed proteins has also been a prominent
test for cultivar identification in crops.
However,
this data does not provide significant discrimination and estimates
of genetic distance that can be useful in helping to provide PVP or
that can be effective in varietal identification. For most species
DUS testing only relies on the comparison of morphological traits
but this is expensive and time consuming. On the other hand these
cytogenitical and biochemical techniques detect only a limited degree
of polymorphism, sensitive to environmental and developmental variation.
Number of markers are also limited and discrimination between different
genotypes is not always possible.
Classical
phenotypic methods of identification are not always sufficient to
solve these problems because of instability of morphological characters
as well as an inability to use such information for identification
at juvenile stages or of isolated plant parts. Therefore, the technologies,
which utilise nuclear DNA and allow complete sampling of the genome,
became useful as a discriminating tool.
DNA
markers and its advantages
DNA
markers have been successfully applied in cultivar identification,
controlling seed purity of hybrids, checking the genetic relatedness
between cultivars, for reducing the reference varieties for comparison
and improving the comparisons of morphological traits. The term DNA
fingerprinting was first used by Alec Jeffrey (1985) to describe the
DNA fragments generated after electrophoratic separation of genomic
DNA by multilocus probes.
Different
types of DNA markers available:...
contd...
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