(callus v.
seedling root)
(callus v.
immature embryo)
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DArT, or diversity arrays technology, enables researchers to analyze plant and animal genomes with no prior DNA sequence knowledge of the organism(s) being investigated. Useful traits in agricultural plants and animals are genetically determined. For most crops, genetic improvement in the last fifty or more years has delivered gains in productivity and improvement in product quality. Much breeding has relied on phenotypic evaluation of individuals and families, with little knowledge of the fundamental genetic changes being incorporated. Genetic fingerprints (genotype) for any individual organism can now be determined by a variety of methods, and the relationship between the genotype and the phenotypic value of the individual can be established. Efficient genotyping technologies are going to play an increasing role in future breeding. However, widespread use of current genotyping technologies is limited by intellectual property constraints, process variation, and high cost per marker.
Current technologies to determine genetic fingerprints have other limitations. Full genome coverage requires the identification of a large number of polymorphic markers. With most current technologies this is a step by step approach that is labor intensive and plagued by process variation. Yes, complete genome sequencing is being done for certain large-market and model crops, including rice, but not for many small market crops and species important in conservation. Even once markers are identified, the cost of scoring most markers ("genotyping") is high and process variation is again a concern. We believe that it should be possible to use high throughput genotyping to characterise many loci for their linkage to useful traits, information on genetic diversity, and tools to recombine that diversity productively. To remove some of these limitations, and to make genotyping efficient and affordable even for conservation purposes and crops where limited resources are available, Diversity Arrays™ Technology was developed at CAMBIA. For throughput and reproducibility it compares favorably with similar technologies. DArT was initially used to detect a large number of genetic differences between varieties of rice and wheat (see publications section), but the technology is expected to be applicable to nearly all plant and animal species.
The patented technology is being maintained available for non-exclusive licensing to any entity. For more information about these agreements, e-mail us. A complete (or "genome-wide") genetic fingerprint of an individual can be useful in the following ways, among others:
Please enter the DArT Technology Forum to learn more or contribute to the exploration of this technology.
Note: This technology is available for use to anyone that agrees to the improvement-sharing conditions of a royalty-free BiOS Genetic Resources Indexing Technology License.
The process involves the development of a genotyping
array.
Genotypes for each DArT marker are then scored using hybridisation with the genotyping array. This can be done in a variety of different ways. In the most common implementation, the genomic representation fragments are stained with a dye that fluoresces green, while vector sequences are stained with a dye that fluoresces red, so that image analysis after the hybridisation, wash and scan can be done by comparison of the intensity of green and red fluorescence as in a typical expression microarray. The outcome is the genotype of the sample: a list of array features (spots) hybridising to the sample. The genotype is a table showing presence (1) or absence (0) for each marker. Any standard genetic analysis software can then be used to analyse the data.
The first example of Diversity Arrays was published by Jaccoud et al., Nucleic Acids Research in February 2001, applied to rice, and the genome complexity reduction technique was derived from AFLP®. For most applications, this would not provide sufficiently complex representations, so we are interested in improvements. One of the major purposes of an open source license is to provide for grantback of improvements to all licensees, not just to the owner or the improver of the technology. Although CAMBIA does not require BiOS licensees to pay royalties, all licenses to DArT contain open source provisions that are required for compliance to maintain the license.
Note: This technology is available for use to anyone that agrees to the improvement-sharing conditions of a royalty-free BiOS Genetic Resources Indexing Technology License.
In order to
detect genetic differences between several cultivars of a crop species the first
step in the DArT technique is to extract DNA from the plants of interest.A
portion of DNA from each cultivar is mixed and the long strands of DNA within
the mixture are then cut into smaller fragments using a selection of enzymes
that target specific regions along the length of the DNA. The result is a pool
of DNA sections of varying sizes, which are then processed through several steps
to produce multiple copies of the smaller fragments. This is called a
'representation' and it has a reduced complexity as compared with the original
genetic material. Eventually, these fragments are placed as tiny spots onto a
batch of identical glass slides using a microarrayer machine.
To determine the genetic differences between any two cultivars of a plant, the second part of the technique involves preparing a 'representation' from each cultivar as before, labelling the DNA with a fluorescent dye and then binding the labelled DNA onto two of the microarrayed glass slides. At this stage, the labelled DNA samples bind only to matching DNA spots on the glass slides, resulting in a signal that can be read and measured by a scanner machine. The result for each cultivar will be different, depending on which spots the DNA binds to, and these differences show the degree of diversity between the two cultivars.
Finally, the slides are scanned for signals generated from each DNA spot and these are converted into binary scores of 0's and 1's. The data is compiled, analysed and managed using the world-class DArTsoft and DArTdb computer package, which was developed and refined in-house to process the large amount of data generated by the technique.
A poster entitled "Diversity Arrays Technology: high-throughput and low-cost whole genome genotyping in plants", presented on behalf of CAMBIA at the 7th International Congress of Plant Molecular Biology summarises the results of projects on a monocot (barley) and a dicot (cassava).
Application of the DArT technology to genetic mapping and diversity analyses of barley is further described in "Diversity Arrays Technology (DArT) for whole-genome profiling of barley," in the Proceedings of the National Academy of Science.
Improvements for the application of Diversity Arrays technology to many of the non-sequenced cultivars of rice have been made by Sujin Patarapuwadol, a CAMBIA PhD student funded by the Rockefeller Foundation. This work is currently being written up.
Improvements to use of DArT technology for apple - a long-lived horticultural crop - have also been made and will be published on this website as soon as all necessary permissions have been obtained.
A report submitted by a licensee to the Generation Challenge Program Workshop in January 2006 discusses the use of DArT in whole genome profiling of crops including its application to sorghum, banana/plantain and coconut palm.
A paper has been published by Akbari M, Wenzl P, Vanessa C, Carling J, Xia L, Yang S, Uszynski G, Mohler V, Lehmensiek A, Kuchel H, Hayden MJ, Howes N, Sharp P, Rathmell B, Vaughan P, Huttner E, Kilian A (2006) Diversity Arrays Technology (DArT) for high-throughput profiling of the hexaploid wheat genome.
Development of improved Eucalyptus genotypes involves the routine identification of breeding stock and superior clones. Currently, microsatellites and random amplified polymorphic DNA markers are the most widely used DNA-based techniques for fingerprinting of these trees. While these techniques have provided rapid and powerful fingerprinting assays, they are constrained by their reliance on gel or capillary electrophoresis, and therefore, relatively low throughput of fragment analysis. In contrast, recently developed microarray technology holds the promise of parallel analysis of thousands of markers in plant genomes. The aim of this study was to develop a DNA fingerprinting chip for Eucalyptus grandis and to investigate its usefulness for fingerprinting of eucalypt trees. A prototype chip was prepared using a partial genomic library from total genomic DNA of 23 E. grandis trees, of which 22 were full siblings. A total of 384 cloned genomic fragments were individually amplified and arrayed onto glass slides. DNA fingerprints were obtained for 17 individuals by hybridizing labeled genome representations of the individual trees to the 384-element chip. Polymorphic DNA fragments were identified by evaluating the binary distribution of their background-corrected signal intensities across full-sib individuals. Among 384 DNA fragments on the chip, 104 (27%) were found to be polymorphic. Hybridization of these polymorphic fragments was highly repeatable (R2>0.91) within the E. grandis individuals, and they allowed us to identify all 17 full-sib individuals. Our results suggest that DNA microarrays can be used to effectively fingerprint large numbers of closely related Eucalyptus trees.
Lezar S, Myburg AA, Berger DK, Wingfield MJ, Wingfield BD. (Nov 2004) Development and assessment of microarray-based DNA fingerprinting in Eucalyptus grandis. Theor Appl Genet 109(7):1329-36.
Department of Genetics, University of Pretoria, Pretoria, 0020, South Africa
Understanding the distribution of genetic diversity within and among individuals, populations, species and gene pools is crucial for the efficient management of germplasm collections. Molecular markers are playing an increasing role in germplasm characterization, yet their broad application is limited by the availability of markers, the costs and the low throughput of existing technologies. This is particularly true for crops of resource-poor farmers such as cassava, Manihot esculenta. Here we report on the development of Diversity Arrays Technology (DArT) for cassava. DArT uses microarrays to detect DNA polymorphism at several hundred genomic loci in a single assay without relying on DNA sequence information. We tested three complexity reduction methods and selected the two that generated genomic representations with the largest frequency of polymorphic clones (PstI/TaqI: 14.6%, PstI/BstNI: 17.2%) to produce large genotyping arrays. Nearly 1,000 candidate polymorphic clones were detected on the two arrays. The performance of the PstI/TaqI array was validated by typing a group of 38 accessions, 24 of them in duplicate. The average call rate was 98.1%, and the scoring reproducibility was 99.8%. DArT markers displayed fairly high polymorphism information content (PIC) values and revealed genetic relationships among the samples consistent with the information available on these samples. Our study suggests that DArT offers advantages over current technologies in terms of cost and speed of marker discovery and analysis. It can therefore be used to genotype large germplasm collections.
Xia L, Peng K, Yang S, Wenzl P, de Vicente MC, Fregene M, Kilian A. (Apr 2005) DArT for high-throughput genotyping of Cassava (Manihot esculenta) and its wild relatives. Theor Appl Genet. 110(6):1092-8.
DArT P/L, GPO Box 3200, Canberra, ACT 2601, Australia.
Understanding the distribution of genetic diversity among individuals, populations and gene pools is crucial for the efficient management of germplasm collections and breeding programs. Diversity analysis is routinely carried out using sequencing of selected gene(s) or molecular marker technologies. Here we report on the development of Diversity Arrays Technology (DArT) for pigeonpea (Cajanus cajan) and its wild relatives. DArT tests thousands of genomic loci for polymorphism and provides the binary scores for hundreds of markers in a single hybridization-based assay. We tested eight complexity reduction methods using various combinations of restriction enzymes and selected PstI/HaeIII genomic representation with the largest frequency of polymorphic clones (19.8%) to produce genotyping arrays. The performance of the PstI/HaeIII array was evaluated by typing 96 accessions representing nearly 20 species of Cajanus. A total of nearly 700 markers were identified with the average call rate of 96.0% and the scoring reproducibility of 99.7%. DArT markers revealed genetic relationships among the accessions consistent with the available information and systematic classification. Most of the diversity was among the wild relatives of pigeonpea or between the wild species and the cultivated C. cajan. Only 64 markers were polymorphic among the cultivated accessions. Such narrow genetic base is likely to represent a serious impediment to breeding progress in pigeonpea. Our study shows that DArT can be effectively applied in molecular systematics and biodiversity studies.
Yang S, Pang W, Ash G, Harper J, Carling J, Wenzl P, Huttner E, Zong X, Kilian A (2006) Low level of genetic diversity in cultivated Pigeonpea compared to its wild relatives is revealed by diversity arrays technology.Theor Appl Genet. 113(4):585-95.
Diversity Arrays Technology (DArT) is a microarray-based DNA marker technique for genome-wide discovery and genotyping of genetic variation. DArT allows simultaneous scoring of hundreds of restriction site based polymorphisms between genotypes and does not require DNA sequence information or site-specific oligonucleotides. This paper demonstrates the potential of DArT for genetic mapping by validating the quality and molecular basis of the markers, using the model plant Arabidopsis thaliana. Restriction fragments from a genomic representation of the ecotype Landsberg erecta (Ler) were amplified by PCR, individualized by cloning and spotted onto glass slides. The arrays were then hybridized with labeled genomic representations of the ecotypes Columbia (Col) and Ler and of individuals from an F2 population obtained from a Col × Ler cross. The scoring of markers with specialized software was highly reproducible and 107 markers could unambiguously be ordered on a genetic linkage map. The marker order on the genetic linkage map coincided with the order on the DNA sequence map. Sequencing of the Ler markers and alignment with the available Col genome sequence confirmed that the polymorphism in DArT markers is largely a result of restriction site polymorphisms. Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00438-005-1145-6
Alexander H. J. Wittenberg, Theo van der Lee, Cyril Cayla, Andrzej Kilian, Richard G. F. Visser and Henk J. Schouten. (August 2005) Validation of the high-throughput marker technology DArT using the model plant Arabidopsis thaliana. Molecular Genetics and Genomics 274(1): 30-39. Department of Plant Sciences, Laboratory of Plant Breeding, The Graduate School Experimental Plant Sciences, Wageningen University and Research Centre, P.O. Box 386, 6700 AJ Wageningen, The Netherlands.
Molecular marker technologies are undergoing a transition from largely serial assays measuring DNA fragment sizes to hybridization-based technologies with high multiplexing levels. Diversity Arrays Technology (DArT) is a hybridization-based technology that is increasingly being adopted by barley researchers. There is a need to integrate the information generated by DArT with previous data produced with gel-based marker technologies. The goal of this study was to build a high-density consensus linkage map from the combined datasets of ten populations, most of which were simultaneously typed with DArT and Simple Sequence Repeat (SSR), Restriction Enzyme Fragment Polymorphism (RFLP) and/or Sequence Tagged Site (STS) markers.
The consensus map, built using a combination of JoinMap 3.0 software and several purpose-built perl scripts, comprised 2,935 loci (2,085 DArT, 850 other loci) and spanned 1,161 cM. It contained a total of 1,629 'bins' (unique loci), with an average inter-bin distance of 0.7 +/- 1.0 cM (median = 0.3 cM). More than 98% of the map could be covered with a single DArT assay. The arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. The locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. The distribution of loci along chromosomes indicated centromeric suppression of recombination in all chromosomes except 5H. DArT markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. On the average, 14 +/- 9 DArT loci were identified within 5 cM on either side of SSR, RFLP or STS loci previously identified as linked to agricultural traits. This barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying DArT markers in molecular breeding schemes. The study also highlights the need for improved software for building consensus maps from high-density segregation data of multiple populations.Wenzl P, Li H, Carling J, Zhou M, Raman H, Paul E, Hearnden P, Maier C, Xia L, Caig V, Ovesna J, Cakir M, Poulsen D, Wang J, Raman R, Smith KP, Muehlbauer GJ, Chalmers KJ, Kleinhofs A, Huttner E, Kilian A. (2006) BMC Genomics 7:206
See page describing improvements that have already been made by our licensees for specific crops.
We anticipate that this BioForge project will help others make the technology more cost-effective for use in a variety of settings, and improve accuracy and ease of analysis.
Your suggestions and experience with any of the following, and any other ideas, are welcome in our Discussion Forum.
Substrates: You may have ideas or data to support implementation of substrates that will be cheaper without sacrificing the 10,000+ spotting capability of the glass slides currently in use.
Platforms: The microarrayer currently used to provide DArT genotyping services is the single most expensive piece of machinery involved in the process; it alone makes DArT prohibitively expensive for many researchers. You may have ideas or data to support implementation of more accessible platforms for the technology.
Imaging: The fluorescent dyes and imaging techniques used to locate polymorphisms and points of hybridization are capital-intensive components of DArT. More affordable variations of these techniques or cheapter alternatives will make the technology much more accessible.
Software and Data Analysis: This is a prime area for improvements in implementing off-the-shelf software for color conversion and data analysis more useful, and more robust statistical methods and analysis used to evaluate polymorphism data.
All licensees commit to an ongoing refinement and improvement of the Diversity Arrays technology; the online collaboration and sharing of methodologies and techniques will help to expedite implementation of these improvements by the community.
The
application of DArT to the human genome is an interesting frontier. The
technology is currently being improved for use in the mouse genome (Ph.D.
project by Damian Jaccoud). The complexity and
redundancy of the genome may create challenges, but these are far outstripped by
the potential discoveries and human health improvements that may result.
Another major potential application of DArT in improving human health is the identification of pathogens in a wide variety of settings. For example, DArT might be usable to determine the presence of pathogenic intestinal bacteria, leading to more rapid treatment for illnesses of the digestive system.
To learn more about DArT or to contribute to the discussion of DArT in public health and medicine, visit the forum.
Dr. Steve Hughes, professor at the Center for Genomics in Society at Exeter University, led a project using DArT to examine the genetic diversity of a protected otter population in England. During the 1950s, the otter population in the UK was decimated by the profligate use of organo-chlorine pesticides. Since the level of pesticide application began its decline, anecdotal evidence suggests a resurgence in the otter population. If the population has in fact recovered, it may serve as a model for the recovery of other top-level predators.
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(Otter Photo courtesy of US NOAA) |
Dr. Hughes and his team collected DNA from individual otter feces (spraint) and analyzed them to determine the number of otters present in the system. Although the method they were using was capable of differentiating otter DNA from the DNA of prey, and of distinguishing males from females, it was slow, tedious, and provided a level of information that was ultimately insufficient, particularly in regard to polymorphic data from the samples.
DArT looks to be a better option for the research team. It promises to provide the low-cost, high throughput capabilities the researcher team seeks, while simultaneously enabling them to analyze a much greater level of genetic data. Preliminary evidence suggested that DArT could be used to identify potentially polymorphic representatives within the otter population, and if this is true it will enable researchers to analyze polymorphic data on a much larger scale than the method used previously. This information may provide further insight into the population's genetic diversity, mating patterns, and other behavioral traits.
Currently, Dr. Hughes is looking for other investigators who are interested in applying DArT to analyses of biodiversity. To learn more about DArT or to contribute to the discussion of DArT and Biodiversity, visit the DArT forum.
The Diversity Arrays or DArT technology was developed in CAMBIA laboratories, then funded by revenues from licensing the E.coli gusA gene for plant research. As with the GUS technology, CAMBIA committed to making the DArT technology widely available to all who will agree to maintain open access to the capability to use and improve it, regardless of ability to pay.
Diversity Arrays were developed and patent claims were structured in order to provide a work-around to some of the many barriers to wide use of other types of genomic marker technology. Some of these barriers include high costs per marker and multiple patent rights owned by licensors who charge high fees or who do not make licenses available.
The DArT technology, in contrast, has been made available through open source provisions in agreements available to everyone who agrees to share. Since publication, most of the patent applications have been abandoned in order to make all the data and uses available within the public domain.
A proof of concept paper using rice was published in 2001 by the CAMBIA-based team that developed this technology:
Jaccoud D, Peng K, Feinstein D, Kilian A, "Diversity Arrays: a solid state technology for sequence information independent genotyping". Nucleic Acids Research 29(4): 25-31
Andrzej Kilian, the inventor on the patent applications, was a principal scientist at CAMBIA, who before taking up a full-time position in the private sector to commercialise DArT technology, was a co-supervisor of several CAMBIA-based Ph.D. students who have been doing further work to apply DArT technology to species such as rice. Findings from this research will be added to the Improvements pages whenever further publications are prepared and made available. The first author of this foundation publication, Damian Jaccoud, has gone on to contribute further work on DArT technology in his Ph.D. thesis. Kaiman Peng worked at CAMBIA under a fellowship from the Rockefeller Foundation, the support of which we gratefully acknowledge.
Farmers and plant breeders in Australia have supported more development of DArT technology. This has been made possible through proposals funded to CAMBIA by the Grains Research and Development Corporation (GRDC), the Rural Industries Research and Development Corporation (RIRDC), Horticulture Australia, and Monticello Research.
Many of the patent applications were purposely abandoned after publication, to allow the technology into the public domain. Subject to the option of the inventors there are still patents in a few countries, which may be used under non-exclusive open source licenses. The "open source" provision is intended to allow all users of the technology to reap the benefit of each others' use and improvement of the technology.
Additional research using DArT technology has been reported by groups based in New Zealand, the Netherlands, Norway, Austria, Sri Lanka, Brasil, Mexico and South Africa. It has been used on plant species ranging from barley to Eucalyptus. DArT technology has also been used in studies not involving plants, including studies of otters and an important pathogen of banana.
DArT Pty. Ltd. is a private company founded by former CAMBIA scientist Andrzej Kilian. Although CAMBIA and other private sources and government funding supported and subsidised the founding of this private company, DArT Pty. Ltd. is no longer related directly to CAMBIA. We are keen to see businesses like this succeed in delivering technology that will be useful.
In summary, in return for permission to use the technologies, a licensee institution agrees to allow and encourage its employees and students to post any improvements made to the technologies on a public website. The licensee institution also agrees not to assert any intellectual property rights to the improvements against other licensees.
All licensees that comply with these license terms are allowed to use the technology for research, public good, and/or commercial product development.
Royalty-free licenses are available to both for-profit and non-profit entities. CAMBIA believes that hunger in many parts of the world can be alleviated not only by providing tools to public good researchers in those communities, but also by removing barriers to micro, small and medium enterprise.
IBM, Nokia, Sun and other IT companies have shown that hardware and software technology can be non-exclusively and open-source licensed and simultaneously generate profitability in a for-profit company. DArT technology provides an example of our attempt to support open-source business models using wet science rather than IT. CAMBIA has already non-exclusively licensed its patents on DArT technology to private companies, collaborations, and research institutes to develop DArT genotyping services for a variety of crops including rice, barley, wheat and apple.
Anyone who agrees to the terms of the license is free to develop and use DArT technology and services, for research or to make a profit.
The information contained in this page was believed to be correct at the time it was collated. New patents and patent applications, altered status of patents, and case law may have resulted in changes in the landscape. CAMBIA makes no warranty that it is correct or up to date at this time and accepts no liability for any use that might be made of it. Corrections or updates to the information are welcome, please send an email to info@bios.net.
