Molecular Markers in Genetic Purity Testing and Varietal Identification
Authors: Ambika Rajendran1 and Dhandapani Raju2
1ICAR-Indian Institute of Maize Research (IIMR), Pusa Campus
2 Scientist, Division of Plant Physiology, ICAR-Indian Agricultural Research Institute (IARI), Pusa Campus


Genetic purity refers to the percentage of contamination by seeds or genetic material of other varieties or species. Genetic purity test is necessary for seed certification of different categories in seed multiplication chain. Contamination due to pollen shedders and physical admixtures during processing causes impurity. Grow-out test (GOT) is often practiced by researchers in detecting off types (genetic purity). GOT is time consuming (takes one full growing season for completion), land intensive, tedious and highly vulnerable to human and environmental error. Unambiguous identification of crop varieties and hybrids is essential for their intellectual property right (IPR) protection, prevention of unauthorized commercial use and misuse of brand name. A set of qualitative and quantitative characters known as "descriptors" are currently used for varietal identification in Distinctiveness Utility and Stability (DUS) testing. Quantitative characters interact with the environment misleading the process of variety identification.
Molecular markers facilitate and resolve complications of conventional GOT and DUS testing. Ideal molecular markers are stable, abundant and detectable in plant tissues regardless of growth and differentiation status. Molecular markers methods are being adopted by the researchers for fingerprinting of cultivars, assessment of genetic purity and varietal identification in seed technology and breeding programmes. In genetic purity assessment, molecular markers detect the degree of contamination due to selfing and outcrossing in hybrid seed lot, segregation of genotypes in DUS testing and trait confirmation in transgenics. The goal for variety identification is to obtain a specific/unique pattern for each variety. Such patterning provides protection to varieties in DUS test and characterization of Essentially Derived Varieties (EDVs).


1. Objectives of the study

Choice of marker varies with the specific objective set in a crop. The mating systems in plants are apomixis (clones), selfing (purelines) and crossing. The level of genetic variation among these is low, intermediate and high respectively. The size and structure of the population to be studied depends upon the pollination nature of crop under study. Higher the variation larger is the sample population and vice versa. DNA based methods are more useful and economical in crops with limited genetic diversity and in perennials where morphological characteristics is time consuming. Different types of molecular markers based on mode of transmission and gene action and their specific utility on genetic purity testing and varietal identification is below

Type of marker Utility
bi-parental nuclear, co-dominant markers Identification of selfed female seed in hybrid seed lot
maternal nuclear, dominant markers Trait purity testing, transgenic trait confirmation
maternal organelle, co-dominant markers Identification of isogenic B line contamination in female A line (parental purity testing) in hybrid seed production
paternal organelle inheritance Restorer gene linked parental purity testing in in hybrid seed production


2. Marker system - Technical knowledge, inheritance and level of polymorphism
DNA based markers are either non PCR based (RFLP) or PCR based markers (RAPD, AFLP, SSR, SNP etc.) according to method of analysis. Table below shows the strength and weaknesses of markers.

Features RFLP RAPD AFLP SSR SNP
Abundance Medium Very high Very high High Very high
Type of polymorphism Single base change Single base change Single base change Repeat length change_multiallelic Single base change_bi-allelic nature
DNA quality High Medium High Medium Medium
DNA sequence information Not required Not required Not required Required Required
Level of polymorphism Medium High High High High
Inheritance Co-dominance Dominance Dominance Co-dominance Co-dominance
Reproducibility High Low Medium High High
Technical complexity High Low Medium Low High
Cost High Low Low High High
Species transferability Medium High High Medium Low
Automation Low Medium Medium High High
Disadvantage Insufficient to characterise all released cultivars Non-public
Time consuming interpretation

(Semagn et al., 2006)

Codominant inheritance of markers allows discriminating homozygotic and heterozygotic state of diploid organisms. Owing to low genetic variation in self pollinated crops researchers use dominant markers like RAPD, CAPS, AFLP etc for detecting polymorphism in self pollinated crops where co-dominant markers fails. Researchers do select dominant markers to detect variation in crop plants where there is no sequence information is available. The number of copies of genome influences the selection in marker system to fingerprint genetic material of crops where the ploidy number is high. Selection of dominant marker may negatively influence the test results due to masking of alleles amplified in the respective copies of genome. Polymorphic Information Content (PIC) value provides an estimate of the discriminating power of a marker. It takes into account both the number of alleles at a locus, and the relative frequencies of these alleles in the population under study. It is a tool to determine polymorphism. Availability of annotated whole genome sequence information in crops like rice, maize, soybean and cotton has made the availability of co-dominant marker information available in database viz., www.gramene.org, www.soybase.org, www.cottongen.org irrespective of its pollination nature. Selection of marker for a crop species is also determined by the sequence information of its closely related crop species. Transferability of markers among laboratories, populations, pedigrees and species is helpful for comparative mapping, gene cloning, reduces the cost of genotyping and extends genetic information from model species to more complicated species.

No marker is complete with all criteria. The kind of study to be undertaken determines the choice of molecular marker systems taking into account most desirable properties. Molecular markers should be suitably incorporated in the system of genetic purity and varietal identification to strengthen the seed sector.

References:

1. http://www.upov.int/upovlex/en/conventions/1991/content.html
2. Semagn K, Bjornstad A and Ndjiondjop M N (2006) An overview of molecular marker methods for plants. African J Biotechnol 5 (25) : 2540-2568






About Author / Additional Info:
I am currently Scientist (Plant Breeding) in ICAR-Indian Institute of Maize Research (IIMR) since five years.