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Diversity Arrays Technology, a novel tool for harnessing crop genetic diversity

Peter Wenzl1, 3, Vanessa Caig3, Jason Carling3, Cyril Cayla1, Margaret Evers1, Damian Jaccoud2, Sujin Patarapuwadol2, Grzegorz Uszynski1, Ling Xia3, Shiying Yang2, Eric Huttner1, 2, 3and Andrzej Kilian1, 2, 3

1 Diversity Arrays Technology Pty Limited, 2 CAMBIA, and 3 Triticarte Pty Ltd, GPO Box 3200, Canberra, ACT 2601, Australia.

Abstract

Genetic diversity is the raw material available to plant breeders. By productively recombining genetic diversity, plant breeders have been successfully producing year after year improved cultivars of the major domesticated species used in the world’s diverse agricultural systems. Molecular genetic markers offer a powerful tool to accelerate and refine this process. Existing genetic marker (genotyping) technologies, mostly developed for applications in human health, have been applied successfully to agricultural species too, but their cost remains prohibitive for most agricultural applications. This is particularly true for species for which no molecular data and very limited resources are available. Because of the limitations of existing marker technologies, we have developed Diversity Arrays Technology (DArT), a novel method to discover and score genetic polymorphic markers. DArT is a sequence-independent, high-throughput method, able to discover hundreds of markers in a single experiment. DArT markers are typed in parallel, using high throughput platforms, with a low cost per datapoint. DArT fingerprints will be useful for accelerating plant breeding, and for the characterisation and management of genetic diversity in domesticated species as well as in their wild relatives. We have developed DArT successfully for rice, barley, wheat and cassava and have produced a dedicated data management and analysis package, a key part of the technology, entirely built from Open Source components. We have a high interest in developing partnerships to establish DArT for many species, and we are developing a network model for the delivery of technology to users.

Media summary

A new genotyping technology will make comprehensive genome profiles an affordable and powerful tool to accelerate plant breeding.

Key Words

genotyping, breeding, marker assisted selection.

Whole genome profiling

The creation of novel improved crop varieties is increasingly relying on markers. Molecular genetic markers, based on DNA sequence polymorphism, arenow widely used to complement phenotypic and protein-based markers. Over the past 20 years, DNA-based markers have been established in many agricultural crops. Molecular markers linked to desirable traits have been used to accelerate plant breeding (Ribaut and Hoisington, 1998), for example by replacing phenotypic assays with single-marker assays when possible and cost-effective (Bonnett et al, 2004). Many traits of interest to plant breeders, however, are complex and polygenic. Therefore the creation of an adapted elite variety will increasingly involve the deliberate combination of various genomic regions from many different individuals (Peleman and van der Voort, 2003). Comprehensive knowledge of genetic diversity in the cultivated and wild germplasm – the source of novel genomic regions, novel alleles and novel traits (Xiao et al, 1998; Li et al, 2003) - is very important. Applying molecular markers in this context requires moving from single marker assays to genome-wide marker profiles: genomic fingerprints covering genetic diversity at hundreds of loci. For genetic diversity analysis also, a reliable measure of the differences and the relatedness between individuals will require whole-genome profiling. After a brief review of the limitations of commonly used marker systems we present here our current work on the development of Diversity Arrays Technology (“DArT”), a novel marker system invented by one of us (AK) and particularly suitable for the analysis and application of crop genetic diversity (Jaccoud et al, 2001).

The limitations of existing technologies

Current molecular marker technologies include RFLP, AFLP, SSR (microsatellites) and SNP. All have at least one of the following limitations:

  • The discovery of a sufficiently large number of polymorphic markers to achieve genome coverage is slow because it is a sequential process.
  • Some marker systems are based on sequence information. For many plant species it may not be practical or economical to determine a large amount of genomic sequence, particularly if different alleles have to be identified by sequencing, for example when sources of new alleles are discovered.
  • Once markers are identified, the cost of scoring the markers (“genotyping “) is high and the throughput low. For SSR and SNP markers an assay has to be developed first. This often results in only a fraction of the candidates identified being retained for routine use. Markers are then typically scored one by one (or at a modest level of multiplexing), usually using gel-based systems.

Diversity Arrays Technology

Diversity Arrays Technology (DArT) was developed to provide a practical and cost-effective whole-genome fingerprinting tool. DArT has three key attributes of interest to plant breeders and scientists studying and managing genetic diversity: (a) it is independent from DNA sequence, (b) the genetic scope of analysis is defined by the user and easily expandable, and (c) the method provides for high throughput and low-cost data production.

Figure 1. Principle of DArT

Sequence independence

The discovery of polymorphic DArT markers and their scoring in subsequent analysis does not require any DNA sequence data. This makes the method applicable to all species, regardless of how much DNA sequence information is available for that species. However DArT markers are sequence-ready clones of genomic DNA.

Genetic scope

For each species, the method is developed on the “metagenome”, the pooled genomes from the germplasm of interest to the user. For example, the metagenome may include DNA from the cultivated varieties of a particular region or the lines used in a breeding program. Alternatively, the metagenome may cover the genetic diversity within the entire species and even extend to its wild relatives. Importantly, the diversity surveyed by DArT can be expanded if new individuals with marked genetic differences are incorporated into the analysis at a later stage.

High-throughput, low-cost data production

In DArT, several hundred polymorphic markers are identified in parallel. The efficiency of this marker discovery effort is only dependent on the level of genetic diversity within the species. For example, 5-10% of wheat and barley DArT clones and 25-30% of cassava DArT clones were polymorphic. The same platform is used for both discovery and scoring of markers, therefore no assay development, apart from consolidating all polymorphic markers into a single genotyping array, is required after the marker discovery. The microarray platform we currently use enables a high level of multiplexing: approximately 5,000 – 8,000 genomic loci are typically surveyed in parallel in single-reaction assays to discover polymorphic markers. For routine genotyping, several hundred markers are typed in parallel using only 50 – 100 ng of genomic DNA. We project that our data production service will soon deliver data for less than 10 Euro cents per datapoint.

Identification of polymorphic DArT markers

A DArT marker is a segment of genomic DNA, the presence of which is polymorphic in a defined genomic representation (see Figure 1). DArT markers are biallelic and behave in a dominant (present vs absent) or co-dominant (2 doses vs 1 dose vs absent) manner.To identify the polymorphic markers, a complexity reduction method is applied on the metagenome, a pool of genomes representing the germplasm of interest. The genomic representation obtained from this pool is then cloned and individual inserts are arrayed on a microarray resulting in a “discovery array”. Labelled genomic representations prepared from the individual genomes included in the pool are hybridised to the discovery array. Polymorphic clones (DArT markers) show variable hybridization signal intensities for different individuals. These clones are subsequently assembled into a “genotyping array” for routine genotyping.

Complexity reduction methods

Many complexity reduction methods can be used (Jaccoud et al, 2001; Peng et al, 2002). A suitable complexity reduction method produces genomic representations that are sufficiently large and contain a sufficient fraction of polymorphic clones to enable the production of a genotyping array containing several hundred markers. Our currently preferred method is based on digestion of genomic DNA with PstI and a frequent cutter, followed by ligation of an adapter to the PstI ends and amplification of PstI fragments using a primer complementary to the adapter. This method was shown to work well in barley, a species with a 5,000-Mbp genome (Wenzl et al, 2004).

Genotyping by hybridisation

For each individual DNA sample being typed, a genomic representation is prepared using a defined complexity reduction method. The representation is labelled and hybridised to a genotyping array, a microarray printed with copies of the DArT markers. The hybridisation signal for each marker is measured and converted into a score.

Technical platform

The platform we are currently using to discover and score polymorphic markers comprises a standard molecular biology laboratory, a microarray printer and scanner, and computer infrastructure to analyse, store and manage the data produced. Platforms other than printed microarrays – for example colour-encoded beads or self-assembling arrays - could be used for the routine typing of samples. These platforms offer good opportunities to reduce further the cost of routine genome profiling.

Software

We have written DArTsoft, a dedicated software for automatic data extraction, which is capable of producing up to 500,000 scores from discovery arrays in less than two hours. With this sort of throughput, sample tracking and data management becomes essential. We are building DArTdb, a laboratory information management system for barcode-facilitated sample tracking, data storage and data management. As a matter of principle we only use Open Source components for all our DArT-related software products.

Current status of DArT development

Barley and wheat

Our work on barley has resulted in the identification of approximately 1000 polymorphic markers from two different genomic representations. A DArT genetic map has been built for a population of double haploid plants derived from a cross between cultivars Steptoe and Morex (Wenzl et al, 2004). We have similarly identified several hundred DArT markers in wheat and are currently building genetic maps of wheat populations from crosses of interest to Australian wheat breeders. Triticarte Pty Ltd, a joint- venture between the Cooperative Research Centre for Value Added Wheat and Diversity Arrays Technology Pty Limited, is now delivering whole-genome profiles of wheat and barley.

Other species

We have identified several hundreds of DArT markers in rice and are developing a genotyping tool in collaboration with the Australian rice industry. We are currently establishing the technology on cassava, apple, pigeonpea, sorghum, chickpea, sugarcane and qinoa, in all cases in partnership with interested users. Together with colleagues from Plant Research International we also established DArT for the model species Arabidopsis thaliana (Wittenberg et al, 2004).

Applications of DArT markers

DArT markers can be used as any other genetic marker. With DArT, comprehensive genome profiles are becoming affordable for virtually any crop, regardless of the molecular information available for the crop. DArT genome profiles are very useful for the recognition and management of bio-diversity, for example in germplasm collections. In plant breeding, DArT genome profiles enable breeders to map QTL in one week, thereby allowing them to focus on the most crucial factor in plant breeding: reliable and precise phenotyping. Once many genomic regions of interest are identified in many different lines, DArT profiles accelerate the introgression of a selected genomic region into an elite genetic background (for example by marker-assisted backcrossing). Furthermore DArT profiles can be used to guide the assembly of many different regions into improved varieties. For that purpose, dense genome cover is essential in order to follow many regions simultaneously. Because of the large number of lines to be typed, high throughput and affordability are critical factors in this context.

A new model for technology delivery

We have initiated the establishment of a network of DArT users, who will contribute their scientific expertise and resources to develop and improve the technology further. Key requirements to join the network are:

  • willingness to share improvements, and
  • acceptance that financial rewards from the delivery of DArT services will not be derived from access to protected IP but from providing efficient “value for the money” services to customers.

The growing success of the Open Source model in the software industry may provide some guidance towards establishing a sustainable system for open access biotechnology, where competition would take place, and profits would be made, at the level of products and services. In this context, we hope that providing DArT services will be a sustainable activity for our organisation and its partners, allowing us to develop and deliver improved genome profiling methods and to apply them to biological research and crop improvement.

Acknowledgments

The development of DArT has been supported by CAMBIA, Rockefeller Foundation, GRDC (Australia), CRC for Value Added Wheat, Rural Industries Research and Development Corporation (Australia), Horticulture Australia, Monticello Research Australia, Australian Wool Innovation, Gardiner Foundation, Australian federal government Biotechnology Innovation Fund, and the government of the Australian Capital Territory.

References

Bonnett, D.G., Rebetzke, G.J. and Spielmeyer, W. (2004) Strategies for efficient implementation of molecular markers in wheat breeding. Molecular Breeding, in press.

Li, Z.K., Fu, B.Y., Gao, Y.M., Xu, J.L., Vijayakumar, C.H.M., Ali, J., Lafitte, R., Ismail, A., Yanagihara, S., Zhao, M.F., Domingo, J., Maghirang, R., Hu, F.Y. and Zhao, X. Q. (2003) Discovery and exploitation of “hidden” genetic diversity in germplasm collections for genetic improvement of abiotic stress tolerances in rice. XIX International Congress of Genetics, Melbourne 6-11 July 2003, www.genunet1.org/IGC2003/abstracts/30MinSpeakers-HTML/Li,%20Zhi-Khang.htm

Peleman, J.D. and van der Voort, J.R. (2003) Breeding by design. Trends in Plant Science 8(7), 330-334.

Peng, K.M., Jaccoud, D., Kudrna, D., Cho, Y.G. and Kilian, A. (2002). Diversity Array Technology (DArT) applications to plant and animal genomics. Plant and Animal Genomes X Conference, San Diego, 12-16 January 2002, www.intl-pag.org/pag/10/abstracts/PAGX_W180.html

Ribaut J.M. and Hoisington, D.A. (1998) Marker-assisted selection: new tools and strategies. Trends in Plant Science 3, 236-239.

Wenzl, P., Carling, J., Kudrna, D., Jaccoud, D., Huttner, E., Kleinhofs, A. and Kilian, A. (2004) Diversity Arrays Technology (DArT) for whole genome-profiling of barley. Proc. Natl. Acad. Sci. USA, in print.

Wittenberg, A.H.J., Kilian, A. and Schouten, H.J. (2004) DArT™, a promising genetic fingerprinting technology. Plant and Animal Genomes XII Conference, San Diego, 10-14 January 2004, www.intl-pag.org/12/abstracts/W36_PAG12_168.html

Xiao J., Li J., Grandillo S., Ahn S.N., Yuan L., Tanksley S.D. and McCouch S.R. (1998) Identification of trait-improving quantitative trait loci alleles from a wild rice relative, Oryza rufipogon. Genetics 150(2), 899-909.

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