DNA Barcoding and its Applications in Agriculture
Authors: DHARA SAVSANI¹, CHANDANI HIHOR ², CHANDNI PATEL ^3
1 Ph.D. Scholars, Dept. of Agricultural Biotechnology, Anand Agricultural University, Anand.
² Senior Research Fellow, Dept. of Animal Genetics and Breeding, College of Veterinary and Animal Husbandry, Anand Agricultural University, Anand.
³ Junior Research Fellow, Gujarat State Biotechnology Mission, Gandhinagar.


DNA Barcoding is a new molecular tool with great potential for easy identification of different species and assigning them to specific taxa.

DNA barcodes consist of a standardised short segments of a gene sequence that in principle evolve fast enough to differentiate species, but have flanking regions that are sufficiently conserved to enable the barcode region to be serviced by universal primers for all species on the planet. Translating this barcode region involves choosing one or a few standard loci that can be sequenced routinely and reliably in very large and diverse sample sets, resulting in easily comparable data which enable species to be distinguish from one another. DNA barcodes have only four alternate nucleotides at each positions, but the string of sites available for inspection is huge.

Criteria for selecting candidate barcode region

I. The DNA barcode regions should be highly conserved priming sites and highly reliable DNA amplification and sequencing
II. The target DNA region should be short to allow easy amplification of DNA
III. The target DNA region should contain enough phylogenetic information to easily assign species to its taxon
IV. Ability to identify or define interspecific sequence differences
V. Ability to distinguish intraspecific sequence differences

DNA Barcoding Resources

• Consortium for the Barcode of Life (CBOL) http://www.barcodeoflife.org in2004
• The Barcode of Life DataSystem (BOLD) International Barcode of Life (iBOL) in 2005
• http://www.ibol.org http://www.boldsystems.org in 2010

Barcoding, an advancement over taxonomy

• Barcoding can identify the species from bits and pecies. It will help reconstruct food cycles by identifying fragments in stomachs and assist plant science by identifying roots sampled from soil layers
• Barcoding can identify a species in its many forms, from eggs and seed, through larvae and seedlings, to adults and flowers
• Barcoding can distinguish among species that look alike, uncovering dangerous organisms masquerading as harmless ones and enabling a more accurate view of biodiversity
• A library of digital barcodes will provide an unambiguous reference that will facilitate identifying species invading and retreating across the globe and through centuries
• Foreseeing millions more species to go, scientists can equip themselves with barcoding to speed identification of known organisms and facilitate rapid recognition of new species
• It will make possible identification of species whether abundant or rare, native or invasive, engendering appreciation of biodiversity locally and globally
• Barcoding newly discovered species will help show where they belong among known species, sprouting new leaves on the tree of life
• Compiling the library of barcodes begins with the multimillions of specimens in museums, herbaria, zoos and gardens, and other biological repositories. The spotlight that barcoding shines on these institutions and their collections will strengthen their ongoing efforts to preserve Earth's biodiversity
• Compiling a library of barcodes linked to vouchered specimens and their binomial names will enhance public access to biological knowledge, helping to create an on-line encyclopedia of life on Earth, with a web page for every species of plant and animal

Microcoding

This system utilizes the fully sequenced genes, which are studied to find a small thermodynamically stable and non-hairpin forming areas of high sequence uniqueness that can be used as single stranded oligonucleotide. It can be developed as a second step in DNA barcoding procedures for any groups of organisms.

Benefits of Barcoding

• DNA barcoding can speed up identification of new species
• DNA barcodes can be linked to readily observable morphological characters
• Applied taxonomic research areas will benefit from barcoding such as disease vectors, agricultural pests, invasives, using minimal samples, damaged specimens, gut contents, droppings
• Food adulteration and forensic science

Case Studies:

Gillian et al., (2011) determined the parentage ofVanda Miss Joaquim by the sequence analysis of matK and rbcL barcodes. The maximum number of nucleotides base variation between samples V. hookeriana and V. teres var. andersonii was eight base substitutions. From the nucleotide and protein sequence alignment, it was apparent that sequence ofV, teres var. andersonii was identical to the sequence of Vanda Miss Joaquim.

Vischi et al., (2006) assessed the variability in wild sunflower species by trnh-psbA intergenic spacer. Intraspecific variation inHelianthus argophyllus was observed by the presence of SNPs andindels. Interspecific variation in H. annuus, H. argophyllus, H. debilis and H. tuberosus was addressed by the presence of SNPs, indels and the number of repeats of the poly A motif.

Chen et al., (2010) validated ITS2 as a novel barcode for the authentication of medicinal plant species by six parameter approach. Of which first three parameters were used to characterize interspecific divergence and the other three for the intraspecific variation. ITS2 region possess high interspecific divergence and intraspecific variation due to the short sequence and high resolution.

Madesis et al., (2012) implemented a two locus DNA barcode technique in certain Mediterranean leguminous crops. The chloroplast trnL barcoding region allowed the discrimination of all crop legumines, but the rpoC1 region allowed the discrimination of only 78% of the species which lead to the combined use of the two barcoding regions to allow the discrimination of the legume crops.

Bruni et al., (2010) confirmed the utility of DNA barcoding for plant poisonous species identification. They supported the application of matK as the best plastidial marker and At103 as the most suitable candidate barcode, which allows a good distinction among congeneric species, including toxic and edible species.

Valentini et al., (2010) opined the trnL approach as a suitable method for the study of plant composition in honey. The sequences of the analysed samples were compared to a plant sequence database. An alignment was retrieved if there was at least 98% identity between the query and database sequences, 100% query coverage and 100% subject sequence coverage provided the information of the plant diversity in the Pyrenean and Wild Flower honey composition.


Conclusion:

• Offers alternative taxonomic identification tool for situations in which morphology is inconclusive.
• DNA Barcoding helps in saving time, money and efforts by circumventing process of genetic fingertyping and genotyping for molecular characterization.
• Focus on one or a small number of genes provides greater efficiency for barcoding.
• Potential capacity for high throughput and processing large number of samples.
• Once reference database is established, can be applied even by a non-specialist.

Future Thrust:
• Needs to explore a single barcode locus for plants
• A standard marker region for plants will be a fulcrum to the success of barcoding life on earth
• Newer methods or algorithims for searching the barcode database has to be investigated to clear the ambiguityin species identification
• A life barcode should be devised to spot species, which can also be connected via the Internet to all other forms of biological data
• Refinement in the barcoding technology to identify hybridity and genetic hindrances like linkages, chromosomal aberrations, epistasis, pseudodominance etc.

References:

1. Bruni H., Fabrizio M., Andrea G., Galasso G., Enrico B., Maurizio C. and Massimo L. (2010).
Identification of poisonous plants by DNA barcoding approach. Int. J. Legal Med., DOI 10.1007/s00414-010-0447-3. 5(1): 8613. doi:10.1371.
2. Chen S., Yao H., Jianping H., Chang L., Jingyuan S., Linchun S., Zhu Y., Xinye M., Gao T., Pang X.,
Luo K., Li Y., Xiwen L., Xiaocheng J., Yulin L. and Christine L. (2010). Validation of ITS2Region as a Novel DNA Barcode for Identifying Medicinal Plant Species. PLoS ONE, doi: 10.1371/journal.pone.0008613.
3. Gao T. and Chen S. L. (2009). Authentication of the medicinal plants in Fabaceae by DNA barcoding technique. Planta Med., 75: 417.
4. Gao T., Sun Z., Yao H. and Song J. (2011). Identification of Fabaceae plants using the DNA barcode matK. Planta Med., 77: 92-94.
5. Gao T., Yao H., Song J. and Liu C. (2010). Identification of medicinal plants in the family fabaceae using a potential DNA barcode ITS2. J. Ethnopharmacol. 130: 116-121.
6. Gillian S. W. K. and Tet F. (2011). Parentage determination of Vanda Miss Joaquim (Orchidaceae) through two chloroplast genes rbcL and matK. AoB PLANTS, doi:10.1093/aobpla/plr018
7. P. M., Graham S. W. and Little D. P. (2011) Choosing and Using a Plant DNA Barcode. PLoS ONE, 6(5): 19254. doi:10.1371.
8. Madesis P., Ganopoulos I., Ralli P. and Tsaftaris A. G. (2012). Barcoding the major Mediterranean leguminous crops by combining universal chloroplast and nuclear DNA sequence targets. Mol. Res., 11 (3): 2548-2558.
9. Summerbell R. C., Vesque C. A., Seifert K. A., Bovers M., Fell J. W., Diaz M. R., Boekhout T., DeHoog G.S., Stalpers J. and Crous P.W. (2005). Microcoding: the second step in DNA barcoding. Phil. Trans. R. Soc. B., 360: 1897-1903.
10. Taylor H. R. and Harris W. E. (2012). An emergent science on the brink of irrelevance: a review of the past 8 years of DNA barcoding. Molecular Ecology Resources, doi: 10.1111/j.1755-0998.2012.03119.
11. Valentini A., Christian M. and Pierre T. (2010) DNA Barcoding for Honey Biodiversity. Diversity, (2): 610-617. doi:10.3390/d2040610.
12. Vischi M., Arzenton F., Paoli D., Paselli E., Tomat S. and Olivieri E. (2006). Identification of Wild species of sunflower by a specific plastid DNA sequence. HELIA, (29) 11-18.
13. Wojciechowski M. F., Lavin M. and Sanderson M. J. (2004). A phylogeny of legumes (Leguminosae) based on analysis of the plastid matK gene resolves many well supported subclades within the family. Am. J. Bot., 91: 1846-1862.


About Author / Additional Info:
I am doing my Ph. D. in Plant Molecular Biology and Biotechnology from Anand Agricultural University