Department of BioTechnology

DNA Banking Genetic Interest

Low Cost, Generic Molecular Markers for Breeding and Research

J. L. Karihaloo, Asia-Pacific Consortium on Agricultural Biotechnology, NASC Complex, Pusa, New Delhi
E-mail: j.karihaloo@cgiar.org

The enormous diversity of world's flora and fauna has been the mainstay of human survival and well being. Genetic resources, comprising useful living organisms, fulfil our basic needs of food, shelter and clothing; provide valuable medicines, spices and materials for industrial products; and help in maintaining and ameliorating the environment. Genes available in wild plants and animals are being constantly used by breeders to improve yield, quality and nutritional value of crops and farm animals.

India is fortunate to have a high and varied diversity of flora. The country possesses 11.9% of the recorded

world's plants including 49,219 higher plant species. India is homeland of 167 cultivated species and 329 wild relatives of crop plants. It has about 30,000 to 50,000 indigenous land races of cultivated plants.

Over the last about 30 years, increasing concern is being expressed over the loss of biodiversity due to human and natural factors. Consequently, worldwide efforts are being made towards conservation of wild and cultivated genetic resources. There are two approaches to plant genetic resources conservation: In-situ conservation which refers to the maintenance of plant populations in the habitat where they naturally occur and evolve. Biosphere reserves and heritage sites are examples of in-situ conservation strategy. Ex-situ conservation is done outside the natural habitat or outside the production area, in facilities called genebanks, specially created for this purpose. Methods of ex-situ conservation include storage of seeds in genebanks at subzero temperatures, maintaining in vitro cultures under slow growing conditions and immersion of tissues, embryos or seeds in liquid nitrogen (cryopreservation), or maintenance of whole plants in field genebanks.

DNA banking

Another form of biological resource that offers tremendous opportunities of practical and academic value is the DNA. In fact, the concept of DNA as a genebank resource has emerged out of the revolution in genomic information brought about by the analysis of DNA extracted from numerous plant species in laboratories across the world. DNA may also be available as amplification products of polymerase chain reaction based experiments. Other biotechnology experiments require construction of DNA libraries, i.e. collection of segments of DNA containing several copies of the part of genome. These include clones of cDNA, cosmid, PAC (plasmid-derived artificial chromosomes), BAC (bacterial artificial chromosomes), YAC (yeast artificial chromosomes) etc. All these DNA forms are being envisioned as an important resource since the DNA can be utilized for several applications, viz. characterizing the source material, understanding genetic and evolutionary relationships between taxa, functional analysis of genes, comparative genomics and plant breeding. Thus, while so far DNA samples have been accumulating in molecular biology and biotechnology laboratories as a spin-off of ongoing projects, the realization of its vast potential has prompted the consideration of DNA collections as a genetic resource. DNA bank is a particular type of genetic resource bank that preserves and distributes the DNA samples and provides associated information.

It must be mentioned here that at present we do not have technology to raise plants from DNA, and DNA banks cannot replace conventional seed genebanks, in vitro repositories or cryobanks. Hence, DNA banking is considered a complementary conservation strategy that together with other conservation strategies leads to an optimum and sustainable use of genetic diversity.

DNA storage

DNA is generally extracted from young growing leaves but can also be obtained from seeds in genebanks and herbarium specimens. The quality of DNA extracted depends upon the condition of the specimen before storage, the storage environment and the duration of storage. Standard protocols are available for DNA extraction, which involve removal of other cellular components while maintaining the integrity of DNA. The protocols need to be suitably modified for different species. Commercial DNA extraction kits, though expensive, are highly efficient in yielding good quality DNA. DNA is a highly stable molecule; degradation kinetics models suggest that fully hydrated DNA kept at room temperature takes about 10,000 years to depolymerise into small fragments. However, degradation due to presence of endonucleases and other cellular components in the extracted DNA can considerably hasten the process of degradation. With increasing fragmentation of DNA template, it's utility for providing useful information decreases progressively. Studies suggest that purified DNA dissolved in buffer, stores well up to 1-2 years at 40C, 4-7 years at -180 C and more than 4 years when stored at -800 C. It has been found that purification procedures used to remove degrading agents and PCR inhibitors may shear the DNA and also remove proteins that stabilized DNA tertiary structure. Long-term stability of extracted DNA is not fully studied. However, when dried, the extracted DNA shows greater stability.

An alternative approach is to store cells and tissues rather than purified DNA, which avoids the uncertainties about the stability of DNA. Further, stored cells and tissues have added advantage of providing a continuous supply of DNA and enabling biochemical and molecular studies of living cells. In any case, it is not recommended that a DNA extract will exist in a bank without the original plant sample from which it has been extracted. Depending upon the available facilities, the reference sample may be in the form of a live plant in field repository, a propagule conserved in a genebank which can be recovered into a full plant, or a herbarium specimen.

DNA banks around the world

While DNA extraction is a routine activity of numerous laboratories working in diverse areas of genetics, biochemistry, molecular biology and biotechnology, DNA banking is not widespread. A recent worldwide survey by International Plant Genetic Resources Institute (now Bioversity International) revealed that of the 274 respondents from 77 countries, 51 (21%) stored DNA while the rest did not. The survey revealed that the majority of institutes do not store DNA due to budget constraints, insufficient infrastructure and lack of trained human resources. However, more than half of the above respondents indicated that they would consider DNA storage if the above constraints are removed. Some of the major plant DNA banks already operational in different parts of the world are:

1.	Australian Plant DNA Bank, Lismore, Australia (http://www.biobank.com)
2.	DNA bank, Instituo de Pesquisas, Jardim Botanico de Rio deJaneiro, Brazi (http://www.jbrj.gov.br/pesquisa/div_molecular/bancodna/sobre_ing.html )
3.	Missouri Botanical Garden, Missouri, USA (http://www.welbcenter.org/dna_banking.html)
4.	Royal Botanic Gardens, Kew, Surrey, Great Britain (http://www.kew.org/data/dnaBank/homepage.html)
5.	South African National Biodiversity Institute DNA Bank, Kirstenbosch, South Africa (http://www.sanbi.org/frames/researchfram.html)

The DNA Bank at Royal Botanical Gardens, Kew (U.K) contains nearly 23,000 samples of plant genomic DNA stored at -800 C. The bank has a large collection of orchid DNA samples and samples of rare and endangered species. The DNA extraction protocol includes a standard CTAB-chloroform extraction with ethanol precipitation and washing, followed by cleaning with caesium chloride/ethidium bromide gradient. The samples are clean enough to be stable at ambient temperature for several days, and for about 10 years under -800 C storage. The Missouri Botanical Garden stores dried samples of plant material, usually young leaves, in a walk-in freezer maintained at -200 C. The International Rice Genome Sequencing Project is a consortium of 10 countries devoted to sequencing, functional analysis of genes and isolation of genes for important agronomic traits in rice. The DNA bank of National Institute of Agrobiological Sciences (Ibaraki, Japan) preserves, manages and provides access to the materials generated by the project for use of researchers throughout the world.

Opportunities for DNA banks

There are a number of areas in which DNA banks could make an impact in the near future. Most promising possibilities in this context are:

Germplasm characterization and management: Detailed characterization of germplasm using a combination of phenotypic and molecular markers improves genebank management in several ways. It allows 1) detection of gaps in collections, identification of duplicates and redundancy, 2) provides valuable knowledge about molecular diversity, genetic and evolutionary relationships, and 3) allows identification of unique genotypes of special importance to genebanks and breeders.

Marker-assisted selection: An important new role for genebanks having DNA samples of germplasms is the application of molecular techniques to identify genes controlling specific traits in collections of cultivated species and wild relatives

DNA barcoding: Global efforts are underway to produce DNA barcodes of all the plant species on earth. DNA banks would greatly facilitate such efforts by providing the required species DNA and thus avoiding the need for undertaking expensive and time- consuming collection trips of depleting rare herbarium specimens.

Exchange of genetic resources: It will be a lot easier to exchange genetic resources as DNA samples, rather than seed or vegetative propagules. Transboundary movement of seed and other planting material requires time consuming inspection and certification for freedom from pests and diseases. Exchanging DNA samples, on the other hand, avoids the need for time consuming and costly certification procedures.

A novel method of DNA distribution has been developed recently whereby DNA clones or PCR products are pasted directly on the pages of books for distribution to users. The National Institute of Agrobiological Sciences, Japan in collaboration with RIKEN Institute, Japan has constructed a DNA book for rice containing 32,000 clones. The DNA can be extracted from the paper and analysed for various purposes.

DNA banking could constitute a complementary conservation strategy for safeguarding the genetic diversity of a crop's genepool, especially if combined with in vitro conservation or

cryopreservation. DNA banks can also serve as back up or safety duplicates of the physical seed, field or in vitro collections, in case of catastrophic losses.

Limitations of DNA banks

Though holding lot of promise and potential for future compilation, management, storage and referencing of earths genetic resources DNA Banking is faced with its own set of limitations.

Methodologies: Several species with high concentrations of polysaccharides, proteins, tannins and lipids in cells pose problems in extraction of DNA of acceptable purity. Relatively short life span of DNA is another limitation necessitating frequent replacement of DNA samples.

Plant recovery: DNA banking cannot be considered as a substitute for conventional conservation strategies since technologies for regeneration of plants from stored DNA have not been developed so far.

Resource and policy: The cost of establishing and operating a DNA laboratory can be quite prohibitive for some genebanks due to resource limitations. The ready availability of DNA extraction chemicals and other consumables, liquid nitrogen and unlimited power supply may be a problem at some locations. It is obvious that the use of marker technologies in genebank management requires significant additional funding and policy support.

Intellectual property rights: Material Transfer Agreements (MTA) which regulate the usage and intellectual property rights (IPRs) of material transferred are specially designed for exchange of seed or vegetative propagules and do not consider IPR issues in the event of exchange of DNA samples. Given the concerns in the developing world about the protection of rights on its biodiversity, there is a need for adequate safeguards against the infringement of IPRs while exchanging DNA samples.

Conclusions

It is envisaged that DNA banks would develop as strategic components of genebanks providing basic information for improved genebank management and facilitating germplasm characterization and utilization. They would serve as a reference basis for evolutionary and comparative genomics studies and DNA barcoding. This, however, will require a proactive approach involving policy and financial support for not only establishment and operation of DNA banks, but for capacity building in molecular biology, genomics, bioinformatics, modern genetic resources management and Web-based networking tools.