Publications by authors named "Jason Carling"

20 Publications

  • Page 1 of 1

Diversity analysis of 80,000 wheat accessions reveals consequences and opportunities of selection footprints.

Nat Commun 2020 09 11;11(1):4572. Epub 2020 Sep 11.

Genetic Resources Program, International Maize and Wheat Improvement Center (CIMMYT), Carretera México-Veracruz Km. 45 El Batán, Texcoco, C.P., 56237, Mexico.

Undomesticated wild species, crop wild relatives, and landraces represent sources of variation for wheat improvement to address challenges from climate change and the growing human population. Here, we study 56,342 domesticated hexaploid, 18,946 domesticated tetraploid and 3,903 crop wild relatives in a massive-scale genotyping and diversity analysis. Using DArTseq technology, we identify more than 300,000 high-quality SNPs and SilicoDArT markers and align them to three reference maps: the IWGSC RefSeq v1.0 genome assembly, the durum wheat genome assembly (cv. Svevo), and the DArT genetic map. On average, 72% of the markers are uniquely placed on these maps and 50% are linked to genes. The analysis reveals landraces with unexplored diversity and genetic footprints defined by regions under selection. This provides fertile ground to develop wheat varieties of the future by exploring specific gene or chromosome regions and identifying germplasm conserving allelic diversity missing in current breeding programs.
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http://dx.doi.org/10.1038/s41467-020-18404-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486412PMC
September 2020

Discovery of thermophilic Bacillales using reduced-representation genotyping for identification.

BMC Microbiol 2020 05 13;20(1):114. Epub 2020 May 13.

Institute of Applied Ecology, University of Canberra, Canberra, ACT, 2601, Australia.

Background: This study demonstrates the use of reduced-representation genotyping to provide preliminary identifications for thermophilic bacterial isolates. The approach combines restriction enzyme digestion and PCR with next-generation sequencing to provide thousands of short-read sequences from across the bacterial genomes. Isolates were obtained from compost, hot water systems, and artesian bores of the Great Artesian Basin. Genomic DNA was double-digested with two combinations of restriction enzymes followed by PCR amplification, using a commercial provider of DArTseq™, Diversity Arrays Technology Pty Ltd. (Canberra, Australia). The resulting fragments which formed a reduced-representation of approximately 2.3% of the genome were sequenced. The sequence tags obtained were aligned against all available RefSeq bacterial genome assemblies by BLASTn to identify the nearest reference genome.

Results: Based on the preliminary identifications, a total of 99 bacterial isolates were identified to species level, from which 8 isolates were selected for whole-genome sequencing to assess the identification results. Novel species and strains were discovered within this set of isolates. The preliminary identifications obtained by reduced-representation genotyping, as well as identifications obtained by BLASTn alignment of the 16S rRNA gene sequence, were compared with those derived from the whole-genome sequence data, using the same RefSeq sequence database for the three methods. Identifications obtained with reduced-representation sequencing agreed with the identifications provided by whole-genome sequencing in 100% of cases. The identifications produced by BLASTn alignment of 16S rRNA gene sequence to the same database differed from those provided by whole-genome sequencing in 37.5% of cases, and produced ambiguous identifications in 50% of cases.

Conclusions: Previously, this method has been successfully demonstrated for use in bacterial identification for medical microbiology. This study demonstrates the first successful use of DArTseq™ for preliminary identification of thermophilic bacterial isolates, providing results in complete agreement with those obtained from whole-genome sequencing of the same isolates. The growing database of bacterial genome sequences provides an excellent resource for alignment of reduced-representation sequence data for identification purposes, and as the available sequenced genomes continue to grow, the technique will become more effective.
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http://dx.doi.org/10.1186/s12866-020-01800-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7222431PMC
May 2020

Identification of quantitative trait loci (QTL) controlling resistance to pea weevil (Bruchus pisorum) in a high-density integrated DArTseq SNP-based genetic map of pea.

Sci Rep 2020 01 8;10(1):33. Epub 2020 Jan 8.

Institute for Sustainable Agriculture, CSIC, Córdoba, E-14004, Spain.

Pea weevil (Bruchus pisorum) is a damaging insect pest affecting pea (Pisum sativum) production worldwide. No resistant cultivars are available, although some levels of incomplete resistance have been identified in Pisum germplasm. To decipher the genetic control underlying the resistance previously identify in P. sativum ssp. syriacum, a recombinant inbred line (RIL F) population was developed. The RIL was genotyped through Diversity Arrays Technology PL's DArTseq platform and screened under field conditions for weevil seed infestation and larval development along 5 environments. A newly integrated genetic linkage map was generated with a subset of 6,540 markers, assembled into seven linkage groups, equivalent to the number of haploid pea chromosomes. An accumulated distance of 2,503 cM was covered with an average density of 2.61 markers cM. The linkage map allowed the identification of three QTLs associated to reduced seed infestation along LGs I, II and IV. In addition, a QTL for reduced larval development was also identified in LGIV. Expression of these QTLs varied with the environment, being particularly interesting QTL BpSI.III that was detected in most of the environments studied. This high-saturated pea genetic map has also allowed the identification of seven potential candidate genes co-located with QTLs for marker-assisted selection, providing an opportunity for breeders to generate effective and sustainable strategies for weevil control.
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http://dx.doi.org/10.1038/s41598-019-56987-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949260PMC
January 2020

Identification of potential candidate genes controlling pea aphid tolerance in a Pisum fulvum high-density integrated DArTseq SNP-based genetic map.

Pest Manag Sci 2020 May 8;76(5):1731-1742. Epub 2020 Jan 8.

Institute for Sustainable Agriculture, CSIC, Córdoba, Spain.

Background: Pea (Pisum sativum) is one of the most important temperate grain legumes in the world, and its production is severely constrained by the pea aphid (Acyrthosiphon pisum). Wild relatives, such as P. fulvum, are valuable sources of allelic diversity to improve the genetic resistance of cultivated pea species against A. pisum attack. To unravel the genetic control underlying resistance to the pea aphid attack, a quantitative trait loci (QTL) analysis was performed using the previously developed high density integrated genetic linkage map originated from an intraspecific recombinant inbred line (RIL) population (P. fulvum: IFPI3260 × IFPI3251).

Results: We accurately evaluated specific resistance responses to pea aphid that allowed the identification, for the first time, of genomic regions that control plant damage and aphid reproduction. Eight QTLs associated with tolerance to pea aphid were identified in LGs I, II, III, IV and V, which individually explained from 17.0% to 51.2% of the phenotypic variation depending on the trait scored, and as a whole from 17.0% to 88.6%. The high density integrated genetic linkage map also allowed the identification of potential candidate genes co-located with the QTLs identified.

Conclusions: Our work shows how the survival of P. fulvum after the pea aphid attack depends on the triggering of a multi-component protection strategy that implies a quantitative tolerance. The genomic regions associated with the tolerance responses of P. fulvum during A. pisum infestation have provided six potential candidate genes that could be useful in marker-assisted selection (MAS) and genomic assisted breeding (GAB) after functional validation in the future. © 2019 Society of Chemical Industry.
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http://dx.doi.org/10.1002/ps.5696DOI Listing
May 2020

Short-read fastA files dataset from complexity-reduced genotyping by sequencing data of bacterial isolates from a public hospital in Australia.

Data Brief 2019 Aug 16;25:104273. Epub 2019 Jul 16.

Institute for Applied Ecology, University of Canberra ACT 2601, Australia.

This data article contains short-read sequences (length 30-69 bp) obtained from complexity-reduced genotyping by sequencing (GBS) of 165 samples bacterial isolates from hospital patients in the Australian Capital Territory, between 2013 and 2015. These samples represented 14 bacterial species. Data format is shown as filtered fastA files obtained from an Illumina HiSeq2500 sequencer. The experimental factors of this research used three complexity reduction methods with three combinations of restriction enzymes: I with I, I with II and I with II.
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http://dx.doi.org/10.1016/j.dib.2019.104273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6661468PMC
August 2019

Identification of bacterial isolates from a public hospital in Australia using complexity-reduced genotyping.

J Microbiol Methods 2019 05 17;160:11-19. Epub 2019 Mar 17.

Institute for Applied Ecology, University of Canberra, ACT 2601, Australia.

Bacterial identification methods used in routine identification of pathogens in medical microbiology include a combination approach of biochemical tests, mass spectrometry or molecular biology techniques. Extensive publicly-available databases of DNA sequence data from pathogenic bacteria have been amassed in recent years; this provides an opportunity for using bacterial genome sequencing for identification purposes. Whole genome sequencing is increasing in popularity, although at present it remains a relatively expensive approach to bacterial identification and typing. Complexity-reduced bacterial genome sequencing provides an alternative. We evaluate genomic complexity-reduction using restriction enzymes and sequencing to identify bacterial isolates. A total of 165 bacterial isolates from hospital patients in the Australian Capital Territory, between 2013 and 2015 were used in this study. They were identified and typed by the Microbiology Department of Canberra Public Hospital, and represented 14 bacterial species. DNA extractions from these samples were processed using a combination of the restriction enzymes PstI with MseI, PstI with HpaII and MseI with HpaII. The resulting sequences (length 30-69 bp) were aligned against publicly available bacterial genome and plasmid sequences. Results of the alignment were processed using a bioinformatics pipeline developed for this project, Currito3.1 DNA Fragment Analysis Software. All 165 samples were correctly identified to genus and species by each of the three combinations of restriction enzymes. A further 35 samples typed to the level of strain identified and compared for consistency with MLST typing data and in silico MLST data derived from the nearest sequenced candidate reference. The high level of agreement between bacterial identification using complexity-reduced genome sequencing and standard hospital identifications indicating that this new approach is a viable alternative for identification of bacterial isolates derived from pathology specimens. The effectiveness of species identification and in particular, strain typing, depends on access to a comprehensive and taxonomically accurate bacterial genome sequence database containing relevant bacterial species and strains.
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http://dx.doi.org/10.1016/j.mimet.2019.03.016DOI Listing
May 2019

A High-Density Integrated DArTseq SNP-Based Genetic Map of and Identification of QTLs Controlling Rust Resistance.

Front Plant Sci 2018 15;9:167. Epub 2018 Feb 15.

Institute for Sustainable Agriculture, CSIC, Córdoba, Spain.

, a wild relative of pea is an important source of allelic diversity to improve the genetic resistance of cultivated species against fungal diseases of economic importance like the pea rust caused by . To unravel the genetic control underlying resistance to this fungal disease, a recombinant inbred line (RIL) population was generated from a cross between two accessions, IFPI3260 and IFPI3251, and genotyped using Diversity Arrays Technology. A total of 9,569 high-quality DArT-Seq and 8,514 SNPs markers were generated. Finally, a total of 12,058 markers were assembled into seven linkage groups, equivalent to the number of haploid chromosomes of and . The newly constructed integrated genetic linkage map of covered an accumulated distance of 1,877.45 cM, an average density of 1.19 markers cM and an average distance between adjacent markers of 1.85 cM. The composite interval mapping revealed three QTLs distributed over two linkage groups that were associated with the percentage of rust disease severity (DS%). QTLs and were located in the LGs II and IV respectively and were consistently identified both in adult plants over 3 years at the field (Córdoba, Spain) and in seedling plants under controlled conditions. Whenever they were detected, their contribution to the total phenotypic variance varied between 19.8 and 29.2. A third QTL () was also located in the LGIVand was environmentally specific as was only detected for DS % in seedlings under controlled conditions. It accounted more than 14% of the phenotypic variation studied. Taking together the data obtained in the study, it could be concluded that the expression of resistance to fungal diseases in originates from the resistant parent IFPI3260.
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http://dx.doi.org/10.3389/fpls.2018.00167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818415PMC
February 2018

A high density GBS map of bread wheat and its application for dissecting complex disease resistance traits.

BMC Genomics 2015 Mar 19;16:216. Epub 2015 Mar 19.

International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641, 06600, Mexico City, DF, Mexico.

Background: Genotyping-by-sequencing (GBS) is a high-throughput genotyping approach that is starting to be used in several crop species, including bread wheat. Anchoring GBS tags on chromosomes is an important step towards utilizing them for wheat genetic improvement. Here we use genetic linkage mapping to construct a consensus map containing 28644 GBS markers.

Results: Three RIL populations, PBW343 × Kingbird, PBW343 × Kenya Swara and PBW343 × Muu, which share a common parent, were used to minimize the impact of potential structural genomic variation on consensus-map quality. The consensus map comprised 3757 unique positions, and the average marker distance was 0.88 cM, obtained by calculating the average distance between two adjacent unique positions. Significant variation of segregation distortion was observed across the three populations. The consensus map was validated by comparing positions of known rust resistance genes, and comparing them to wheat reference genome sequences recently published by the International Wheat Genome Sequencing Consortium, Rye and Ae. tauschii genomes. Three well-characterized rust resistance genes (Sr58/Lr46/Yr29, Sr2/Yr30/Lr27, and Sr57/Lr34/Yr18) and 15 published QTLs for wheat rusts were validated with high resolution. Fifty-two per cent of GBS tags on the consensus map were successfully aligned through BLAST to the right chromosomes on the wheat reference genome sequence.

Conclusion: The consensus map should provide a useful basis for analyzing genome-wide variation of complex traits. The identified genes can then be explored as genetic markers to be used in genomic applications in wheat breeding.
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http://dx.doi.org/10.1186/s12864-015-1424-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381402PMC
March 2015

Genome-wide delineation of natural variation for pod shatter resistance in Brassica napus.

PLoS One 2014 9;9(7):e101673. Epub 2014 Jul 9.

Graham Centre for Agricultural Innovation (an alliance between NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga Agricultural Institute, Wagga Wagga, NSW, Australia.

Resistance to pod shattering (shatter resistance) is a target trait for global rapeseed (canola, Brassica napus L.), improvement programs to minimise grain loss in the mature standing crop, and during windrowing and mechanical harvest. We describe the genetic basis of natural variation for shatter resistance in B. napus and show that several quantitative trait loci (QTL) control this trait. To identify loci underlying shatter resistance, we used a novel genotyping-by-sequencing approach DArT-Seq. QTL analysis detected a total of 12 significant QTL on chromosomes A03, A07, A09, C03, C04, C06, and C08; which jointly account for approximately 57% of the genotypic variation in shatter resistance. Through Genome-Wide Association Studies, we show that a large number of loci, including those that are involved in shattering in Arabidopsis, account for variation in shatter resistance in diverse B. napus germplasm. Our results indicate that genetic diversity for shatter resistance genes in B. napus is limited; many of the genes that might control this trait were not included during the natural creation of this species, or were not retained during the domestication and selection process. We speculate that valuable diversity for this trait was lost during the natural creation of B. napus. To improve shatter resistance, breeders will need to target the introduction of useful alleles especially from genotypes of other related species of Brassica, such as those that we have identified.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101673PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4090071PMC
March 2015

Quantitative trait loci in hop (Humulus lupulus L.) reveal complex genetic architecture underlying variation in sex, yield and cone chemistry.

BMC Genomics 2013 May 30;14:360. Epub 2013 May 30.

School of Plant Science, University of Tasmania, Hobart, TAS 7001, Australia.

Background: Hop (Humulus lupulus L.) is cultivated for its cones, the secondary metabolites of which contribute bitterness, flavour and aroma to beer. Molecular breeding methods, such as marker assisted selection (MAS), have great potential for improving the efficiency of hop breeding. The success of MAS is reliant on the identification of reliable marker-trait associations. This study used quantitative trait loci (QTL) analysis to identify marker-trait associations for hop, focusing on traits related to expediting plant sex identification, increasing yield capacity and improving bittering, flavour and aroma chemistry.

Results: QTL analysis was performed on two new linkage maps incorporating transferable Diversity Arrays Technology (DArT) markers. Sixty-three QTL were identified, influencing 36 of the 50 traits examined. A putative sex-linked marker was validated in a different pedigree, confirming the potential of this marker as a screening tool in hop breeding programs. An ontogenetically stable QTL was identified for the yield trait dry cone weight; and a QTL was identified for essential oil content, which verified the genetic basis for variation in secondary metabolite accumulation in hop cones. A total of 60 QTL were identified for 33 secondary metabolite traits. Of these, 51 were pleiotropic/linked, affecting a substantial number of secondary metabolites; nine were specific to individual secondary metabolites.

Conclusions: Pleiotropy and linkage, found for the first time to influence multiple hop secondary metabolites, have important implications for molecular selection methods. The selection of particular secondary metabolite profiles using pleiotropic/linked QTL will be challenging because of the difficulty of selecting for specific traits without adversely changing others. QTL specific to individual secondary metabolites, however, offer unequalled value to selection programs. In addition to their potential for selection, the QTL identified in this study advance our understanding of the genetic control of traits of current economic and breeding significance in hop and demonstrate the complex genetic architecture underlying variation in these traits. The linkage information obtained in this study, based on transferable markers, can be used to facilitate the validation of QTL, crucial to the success of MAS.
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http://dx.doi.org/10.1186/1471-2164-14-360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680207PMC
May 2013

Genomic characterization of DArT markers based on high-density linkage analysis and physical mapping to the Eucalyptus genome.

PLoS One 2012 11;7(9):e44684. Epub 2012 Sep 11.

Plant Genetics Laboratory, EMBRAPA Genetic Resources and Biotechnology, Brasilia, Brazil.

Diversity Arrays Technology (DArT) provides a robust, high throughput, cost-effective method to query thousands of sequence polymorphisms in a single assay. Despite the extensive use of this genotyping platform for numerous plant species, little is known regarding the sequence attributes and genome-wide distribution of DArT markers. We investigated the genomic properties of the 7,680 DArT marker probes of a Eucalyptus array, by sequencing them, constructing a high density linkage map and carrying out detailed physical mapping analyses to the Eucalyptus grandis reference genome. A consensus linkage map with 2,274 DArT markers anchored to 210 microsatellites and a framework map, with improved support for ordering, displayed extensive collinearity with the genome sequence. Only 1.4 Mbp of the 75 Mbp of still unplaced scaffold sequence was captured by 45 linkage mapped but physically unaligned markers to the 11 main Eucalyptus pseudochromosomes, providing compelling evidence for the quality and completeness of the current Eucalyptus genome assembly. A highly significant correspondence was found between the locations of DArT markers and predicted gene models, while most of the 89 DArT probes unaligned to the genome correspond to sequences likely absent in E. grandis, consistent with the pan-genomic feature of this multi-Eucalyptus species DArT array. These comprehensive linkage-to-physical mapping analyses provide novel data regarding the genomic attributes of DArT markers in plant genomes in general and for Eucalyptus in particular. DArT markers preferentially target the gene space and display a largely homogeneous distribution across the genome, thereby providing superb coverage for mapping and genome-wide applications in breeding and diversity studies. Data reported on these ubiquitous properties of DArT markers will be particularly valuable to researchers working on less-studied crop species who already count on DArT genotyping arrays but for which no reference genome is yet available to allow such detailed characterization.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0044684PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3439404PMC
March 2013

Diversity arrays technology: a generic genome profiling technology on open platforms.

Methods Mol Biol 2012 ;888:67-89

Diversity Arrays Technology Pty Ltd, Yarralumla, Canberra, ACT, Australia.

In the last 20 years, we have observed an exponential growth of the DNA sequence data and simular increase in the volume of DNA polymorphism data generated by numerous molecular marker technologies. Most of the investment, and therefore progress, concentrated on human genome and genomes of selected model species. Diversity Arrays Technology (DArT), developed over a decade ago, was among the first "democratizing" genotyping technologies, as its performance was primarily driven by the level of DNA sequence variation in the species rather than by the level of financial investment. DArT also proved more robust to genome size and ploidy-level differences among approximately 60 organisms for which DArT was developed to date compared to other high-throughput genotyping technologies. The success of DArT in a number of organisms, including a wide range of "orphan crops," can be attributed to the simplicity of underlying concepts: DArT combines genome complexity reduction methods enriching for genic regions with a highly parallel assay readout on a number of "open-access" microarray platforms. The quantitative nature of the assay enabled a number of applications in which allelic frequencies can be estimated from DArT arrays. A typical DArT assay tests for polymorphism tens of thousands of genomic loci with the final number of markers reported (hundreds to thousands) reflecting the level of DNA sequence variation in the tested loci. Detailed DArT methods, protocols, and a range of their application examples as well as DArT's evolution path are presented.
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http://dx.doi.org/10.1007/978-1-61779-870-2_5DOI Listing
September 2012

Diversity arrays technology (DArT) markers in apple for genetic linkage maps.

Mol Breed 2012 Mar 15;29(3):645-660. Epub 2011 May 15.

Diversity Arrays Technology (DArT) provides a high-throughput whole-genome genotyping platform for the detection and scoring of hundreds of polymorphic loci without any need for prior sequence information. The work presented here details the development and performance of a DArT genotyping array for apple. This is the first paper on DArT in horticultural trees. Genetic mapping of DArT markers in two mapping populations and their integration with other marker types showed that DArT is a powerful high-throughput method for obtaining accurate and reproducible marker data, despite the low cost per data point. This method appears to be suitable for aligning the genetic maps of different segregating populations. The standard complexity reduction method, based on the methylation-sensitive PstI restriction enzyme, resulted in a high frequency of markers, although there was 52-54% redundancy due to the repeated sampling of highly similar sequences. Sequencing of the marker clones showed that they are significantly enriched for low-copy, genic regions. The genome coverage using the standard method was 55-76%. For improved genome coverage, an alternative complexity reduction method was examined, which resulted in less redundancy and additional segregating markers. The DArT markers proved to be of high quality and were very suitable for genetic mapping at low cost for the apple, providing moderate genome coverage. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9579-5) contains supplementary material, which is available to authorized users.
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http://dx.doi.org/10.1007/s11032-011-9579-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3285764PMC
March 2012

Population genetic analysis and phylogeny reconstruction in Eucalyptus (Myrtaceae) using high-throughput, genome-wide genotyping.

Mol Phylogenet Evol 2011 Apr 16;59(1):206-24. Epub 2011 Feb 16.

School of Plant Science, University of Tasmania, Private Bag 55, Hobart, Tasmania 7001, Australia.

A set of over 8000 Diversity Arrays Technology (DArT) markers was tested for its utility in high-resolution population and phylogenetic studies across a range of Eucalyptus taxa. Small-scale population studies of Eucalyptus camaldulensis, Eucalyptus cladocalyx, Eucalyptus globulus, Eucalyptus grandis, Eucalyptus nitens, Eucalyptus pilularis and Eucalyptus urophylla demonstrated the potential of genome-wide genotyping with DArT markers to differentiate species, to identify interspecific hybrids and to resolve biogeographic disjunctions within species. The population genetic studies resolved geographically partitioned clusters in E. camaldulensis, E. cladocalyx, E. globulus and E. urophylla that were congruent with previous molecular studies. A phylogenetic study of 94 eucalypt species provided results that were largely congruent with traditional taxonomy and ITS-based phylogenies, but provided more resolution within major clades than had been obtained previously. Ascertainment bias (the bias introduced in a phylogeny from using markers developed in a small sample of the taxa that are being studied) was not detected. DArT offers an unprecedented level of resolution for population genetic, phylogenetic and evolutionary studies across the full range of Eucalyptus species.
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http://dx.doi.org/10.1016/j.ympev.2011.02.003DOI Listing
April 2011

A high-density Diversity Arrays Technology (DArT) microarray for genome-wide genotyping in Eucalyptus.

Plant Methods 2010 Jun 30;6:16. Epub 2010 Jun 30.

School of Plant Science and Cooperative Research Centre for Forestry, University of Tasmania, Private Bag 55, Hobart, Tasmania 7001, Australia.

Background: A number of molecular marker technologies have allowed important advances in the understanding of the genetics and evolution of Eucalyptus, a genus that includes over 700 species, some of which are used worldwide in plantation forestry. Nevertheless, the average marker density achieved with current technologies remains at the level of a few hundred markers per population. Furthermore, the transferability of markers produced with most existing technology across species and pedigrees is usually very limited. High throughput, combined with wide genome coverage and high transferability are necessary to increase the resolution, speed and utility of molecular marker technology in eucalypts. We report the development of a high-density DArT genome profiling resource and demonstrate its potential for genome-wide diversity analysis and linkage mapping in several species of Eucalyptus.

Findings: After testing several genome complexity reduction methods we identified the PstI/TaqI method as the most effective for Eucalyptus and developed 18 genomic libraries from PstI/TaqI representations of 64 different Eucalyptus species. A total of 23,808 cloned DNA fragments were screened and 13,300 (56%) were found to be polymorphic among 284 individuals. After a redundancy analysis, 6,528 markers were selected for the operational array and these were supplemented with 1,152 additional clones taken from a library made from the E. grandis tree whose genome has been sequenced. Performance validation for diversity studies revealed 4,752 polymorphic markers among 174 individuals. Additionally, 5,013 markers showed segregation when screened using six inter-specific mapping pedigrees, with an average of 2,211 polymorphic markers per pedigree and a minimum of 859 polymorphic markers that were shared between any two pedigrees.

Conclusions: This operational DArT array will deliver 1,000-2,000 polymorphic markers for linkage mapping in most eucalypt pedigrees and thus provide high genome coverage. This array will also provide a high-throughput platform for population genetics and phylogenetics in Eucalyptus. The transferability of DArT across species and pedigrees is particularly valuable for a large genus such as Eucalyptus and will facilitate the transfer of information between different studies. Furthermore, the DArT marker array will provide a high-resolution link between phenotypes in populations and the Eucalyptus reference genome, which will soon be completed.
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http://dx.doi.org/10.1186/1746-4811-6-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2903579PMC
June 2010

Isolated chromosomes as a new and efficient source of DArT markers for the saturation of genetic maps.

Theor Appl Genet 2010 Aug 4;121(3):465-74. Epub 2010 Apr 4.

Diversity Arrays Technology Pty Ltd, 1 Wilf Crane Crescent, Yarralumla, ACT, 2600, Australia.

We describe how the diversity arrays technology (DArT) can be coupled with chromosome sorting to increase the density of genetic maps in specific genome regions. Chromosome 3B and the short arm of chromosome 1B (1BS) of wheat were isolated by flow cytometric sorting and used to develop chromosome- and chromosome arm-enriched genotyping arrays containing 2,688 3B clones and 384 1BS clones. Linkage analysis showed that 553 of the 711 polymorphic 3B-derived markers (78%) mapped to chromosome 3B, and 59 of the 68 polymorphic 1BS-derived markers (87%) mapped to chromosome 1BS, confirming the efficiency of the chromosome-sorting approach. To demonstrate the potential for saturation of genetic maps, we constructed a consensus map of chromosome 3B using 19 mapping populations, including some that were genotyped with the 3B-enriched array. The 3B-derived DArT markers doubled the number of genetic loci covered. The resulting consensus map, probably the densest genetic map of 3B available to this date, contains 939 markers (779 DArTs and 160 other markers) that segregate on 304 genetically distinct loci. Importantly, only 2,688 3B-derived clones (probes) had to be screened to obtain almost twice as many polymorphic 3B markers (510) as identified by screening approximately 70,000 whole genome-derived clones (269). Since an enriched DArT array can be developed from less than 5 ng of chromosomal DNA, a quantity which can be obtained within 1 h of sorting, this approach can be readily applied to any crop for which chromosome sorting is available.
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http://dx.doi.org/10.1007/s00122-010-1323-8DOI Listing
August 2010

Diversity arrays technology (DArT) for high-throughput profiling of the hexaploid wheat genome.

Theor Appl Genet 2006 Nov 11;113(8):1409-20. Epub 2006 Oct 11.

Triticarte P/L, 1 Wilf Crane Crescent, Yarralumla, Canberra, ACT, 2600, Australia.

Despite a substantial investment in the development of panels of single nucleotide polymorphism (SNP) markers, the simple sequence repeat (SSR) technology with a limited multiplexing capability remains a standard, even for applications requiring whole-genome information. Diversity arrays technology (DArT) types hundreds to thousands of genomic loci in parallel, as previously demonstrated in a number diploid plant species. Here we show that DArT performs similarly well for the hexaploid genome of bread wheat (Triticum aestivum L.). The methodology previously used to generate DArT fingerprints of barley also generated a large number of high-quality markers in wheat (99.8% allele-calling concordance and approximately 95% call rate). The genetic relationships among bread wheat cultivars revealed by DArT coincided with knowledge generated with other methods, and even closely related cultivars could be distinguished. To verify the Mendelian behaviour of DArT markers, we typed a set of 90 Cranbrook x Halberd doubled haploid lines for which a framework (FW) map comprising a total of 339 SSR, restriction fragment length polymorphism (RFLP) and amplified fragment length polymorphism (AFLP) markers was available. We added an equal number of DArT markers to this data set and also incorporated 71 sequence tagged microsatellite (STM) markers. A comparison of logarithm of the odds (LOD) scores, call rates and the degree of genome coverage indicated that the quality and information content of the DArT data set was comparable to that of the combined SSR/RFLP/AFLP data set of the FW map.
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http://dx.doi.org/10.1007/s00122-006-0365-4DOI Listing
November 2006

A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits.

BMC Genomics 2006 Aug 12;7:206. Epub 2006 Aug 12.

Triticarte P/L, PO Box 7141 Yarralumla, Canberra, ACT 2600, Australia.

Background: 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.

Results: 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.

Conclusion: Our 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.
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http://dx.doi.org/10.1186/1471-2164-7-206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1564146PMC
August 2006

Low level of genetic diversity in cultivated Pigeonpea compared to its wild relatives is revealed by diversity arrays technology.

Theor Appl Genet 2006 Aug 15;113(4):585-95. Epub 2006 Jul 15.

DArT P/L, PO Box 7141, Yarralumla, ACT 2600, 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.
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http://dx.doi.org/10.1007/s00122-006-0317-zDOI Listing
August 2006

Diversity Arrays Technology (DArT) for whole-genome profiling of barley.

Proc Natl Acad Sci U S A 2004 Jun 10;101(26):9915-20. Epub 2004 Jun 10.

Center for the Application of Molecular Biology to International Agriculture, G.P.O. Box 3200, Canberra, ACT 2601, Australia.

Diversity Arrays Technology (DArT) can detect and type DNA variation at several hundred genomic loci in parallel without relying on sequence information. Here we show that it can be effectively applied to genetic mapping and diversity analyses of barley, a species with a 5,000-Mbp genome. We tested several complexity reduction methods and selected two that generated the most polymorphic genomic representations. Arrays containing individual fragments from these representations generated DArT fingerprints with a genotype call rate of 98.0% and a scoring reproducibility of at least 99.8%. The fingerprints grouped barley lines according to known genetic relationships. To validate the Mendelian behavior of DArT markers, we constructed a genetic map for a cross between cultivars Steptoe and Morex. Nearly all polymorphic array features could be incorporated into one of seven linkage groups (98.8%). The resulting map comprised approximately 385 unique DArT markers and spanned 1,137 centimorgans. A comparison with the restriction fragment length polymorphism-based framework map indicated that the quality of the DArT map was equivalent, if not superior, to that of the framework map. These results highlight the potential of DArT as a generic technique for genome profiling in the context of molecular breeding and genomics.
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http://dx.doi.org/10.1073/pnas.0401076101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC470773PMC
June 2004
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