Improvements made to DArt™ Technology
Improvements made at CAMBIA:
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.
Improvements by others who've agreed to share:
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.
The following improvements have been published:
Development and assessment of microarray-based DNA fingerprinting in Eucalyptus grandis
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
DArT for high-throughput genotyping of Cassava (Manihot esculenta) and its wild relatives
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.
Low level of genetic diversity in cultivated Pigeonpea compared to its wild relatives is revealed by diversity arrays technology.
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.
Validation of the high-throughput marker technology DArT using the model plant Arabidopsis thaliana
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.
A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits.
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