Development of DNA-based markers for genetic fidelity testing of tissue culture raised plants and for phylogenetic studies.
The National consultation group on certification of tissue culture raised plants sponsored by Department of Biotechnology has proposed that plant certification is essential for import/export material as per the rules/procedures laid down in the Plant Quarantine Order 2003, whose primary goal is to ensure production of virus-free plants of assured quality raised through tissue culture. The need for identification of more region-based centres, especially around the parts of the country and also at locations where there is a hub of tissue culture units, was recognized in view of the large volume of material to be tested, for which Department of Biotechnology has been notified as the Certification Agency under the National Certification System for Tissue Culture Raised Plants (NCSTCP).
In view of the above recommendations and proposals, a referral Centre for the genetic fidelity testing of tissue culture raised plants employing DNA markers was established at CDFD to undertake the true-to-type testing of important crop plants like banana, potato, sugarcane, black pepper and vanilla. As part of the project, we have initiated work for the development, standardization and validation of microsatellite or simple sequence repeats (SSR) markers and amplified fragment length polymorphism (AFLP) techniques to test true-to-typeness of above micropropagated plants. Microsatellites are tandemly repeated motifs of 1-6 bases and are widely distributed throughout the eukaryotic genomes, making them the preferred markers for very-high resolution genetic mapping. SSRs in plants are mostly located in coding regions and rarely in non-coding repetitive regions and are characterized by a high degree of length polymorphism. SSRs have proven to be an extremely valuable tool for genome mapping in many organisms but their applications span over different areas ranging from ancient and forensic DNA sample studies to population genetics and conservation/management of biological resources.
A major challenge for utilizing SSR markers for genetic fidelity testing is the requirement of prior information of genomic DNA sequence for designing primers to the flanking regions of the repeat motifs. In the absence of such information, one has to isolate repeat units de novo from most of the species, as DNA sequences are not yet known for designing the primers. This would entail the construction of a genomic/sub-genomic library of that particular plant species/variety. In the present project, since the entire genomic sequences of potato, banana, sugarcane, black pepper and vanilla are not available in the public domain, SSRs would be isolated from these plants based on the selective hybridization procedure.
Referral Centre for Detection of Genetically Modified Foods Employing DNA-based Markers.
The advent of genetic engineering in agriculture and food production has impacted, not only on the environment and biodiversity, but also the human health. A thorough Biosafety assessment requires not only evaluation of environmental impacts of Genetically Modified Organisms (GMOs), but also assessment of the risks that genetically engineered foods pose for the health of the consumers. The need to monitor and verify the presence and the amount of GMOs in agricultural crops and in products derived thereof has generated a demand for analytical methods capable of detecting, identifying and quantifying either the DNA introduced or the proteins expressed in transgenic plants, because these components are considered as the fundamental constituents.
The methods presently employed by various laboratories worldwide for GMO detection and quantification differ in their reliability, robustness and reproducibility and have different levels of cost, complexity and speed. Also, there is no one universal method that is applicable to all circumstances. In order to obtain comparable results, and hence giving consumers and producers confidence in the testing methods, an urgent need was felt for internationally validated methods, which could serve as reference methods. A referral centre for detection of genetically modified foods employing DNA-based markers was established at CDFD. As part of this project, it is proposed to undertake the GM food detection services employing the protocols developed earlier at CDFD in cotton and rice (whole or crushed seeds). In the research and development component of the project, we plan to develop, standardize, validate and employ DNA-based markers for GM food detection for various crops/varieties containing specific transgenes as envisaged by the Joint Working Group of the Department of Biotechnology and Ministry of Health & Family Welfare, Government of India.
Development of novel strategies/methodologies for enrichment of human DNA from mixtures containing human and non-human DNAs for human identification purposes from forensic samples.
In forensic human DNA testing, it has been a common observation that the DNA extracted from bone and teeth samples of human skeletal remains is scant, heavily degraded/ fragmented and contaminated with non-human DNAs which makes the process of DNA testing difficult. Owing to its tropical environment, which results in robust microbial infestation, obtaining good quality human DNA for body identification by DNA profiling has not been very successful in India. Therefore newer strategies have to be explored to selectively enrich for human DNA from the mixture of DNAs and increase the success rate of DNA profiling tests to circumvent these problems. Mass fatality incidents like natural disasters (earthquakes, tsunamis, flooding and cyclones), or man-made disasters (aircraft cashes, train crashes and derailments, terrorist attacks and building fires) results in a very large number of casualties, DNA testing plays an important role in human remains identification. However, degradation and contamination of DNA extracted from bone and teeth samples with non-human DNA could make that process difficult.
We are exploring the possibility of selectively enriching ("pull down") the human DNA sequences from the mixture of non-human DNAs by employing antibodies specific to human DNA sequences. Subsequently, we would amplify such human genome sequences manifold and the human short tandem repeat (STR)-containing fragments would be "captured" by employing biotinylated oligonucleotides and subjected to STR/mini-STR analysis to unambiguously identify the body parts/ skeletal remains of deceased persons. In cases where the DNA obtained from human skeletal remains is heavily degraded, single nucleotide polymorphism (SNP) markers offer a better solution for human identification purposes as compared to STR technology. Therefore, our research interests are also directed at standardizing the SNP technology for human identification purposes in India.
To study the genetic diversity among various caste and tribal populations of India.
The diverse caste and tribal populations in India offer a rich source of human biologic variation, whose study would aid in our understanding and knowledge of social stratification, human evolution and migration patterns. In India, various studies have been undertaken to assess the genetic diversity among various population groups with varying degrees of success. In order to better understand the human diversity in India, the agricultural and hunting-gathering tribal populations would be sampled from India and the distribution of uni- and bi-parental genetic markers would be studied in among these populations. The knowledge gained by the proposed human genetic variation studies in different populations of India would provide new insights in our understanding of human genetic variability. The data obtained from the studies will be compared with published data from other population groups and regions from rest of the country and the world to ascertain male and female migration patterns during the evolution of native populations and to address questions about patrilocality v/s matrilocality in the course of human pre-history and evolution.
Dr. Madhusudan Reddy Nandineni