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1.  A high density GBS map of bread wheat and its application for dissecting complex disease resistance traits 
BMC Genomics  2015;16(1):216.
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.
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.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1424-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4381402  PMID: 25887001
Consensus map; Genotyping-by-sequencing (GBS); QTL mapping; Rust resistance; Segregation distortion; Wheat
2.  A comprehensive genetic map of sugarcane that provides enhanced map coverage and integrates high-throughput Diversity Array Technology (DArT) markers 
BMC Genomics  2014;15(1):152.
Sugarcane genetic mapping has lagged behind other crops due to its complex autopolyploid genome structure. Modern sugarcane cultivars have from 110-120 chromosomes and are in general interspecific hybrids between two species with different basic chromosome numbers: Saccharum officinarum (2n = 80) with a basic chromosome number of 10 and S. spontaneum (2n = 40-128) with a basic chromosome number of 8. The first maps that were constructed utilised the single dose (SD) markers generated using RFLP, more recent maps generated using AFLP and SSRs provided at most 60% genome coverage. Diversity Array Technology (DArT) markers are high throughput allowing greater numbers of markers to be generated.
Progeny from a cross between a sugarcane variety Q165 and a S. officinarum accession IJ76-514 were used to generate 2467 SD markers. A genetic map of Q165 was generated containing 2267 markers, These markers formed 160 linkage groups (LGs) of which 147 could be placed using allelic information into the eight basic homology groups (HGs) of sugarcane. The HGs contained from 13 to 23 LGs and from 204 to 475 markers with a total map length of 9774.4 cM and an average density of one marker every 4.3 cM. Each homology group contained on average 280 markers of which 43% were DArT markers 31% AFLP, 16% SSRs and 6% SNP markers. The multi-allelic SSR and SNP markers were used to place the LGs into HGs.
The DArT array has allowed us to generate and map a larger number of markers than ever before and consequently to map a larger portion of the sugarcane genome. This larger number of markers has enabled 92% of the LGs to be placed into the 8 HGs that represent the basic chromosome number of the ancestral species, S. spontaneum. There were two HGs (HG2 and 8) that contained larger numbers of LGs verifying the alignment of two sets of S. officinarum chromosomes with one set of S. spontaneum chromosomes and explaining the difference in basic chromosome number between the two ancestral species. There was also evidence of more complex structural differences between the two ancestral species.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-152) contains supplementary material, which is available to authorized users.
PMCID: PMC4007999  PMID: 24564784
Saccharum; Polyploid; Genetic mapping; SNP markers; DArT sugarcane array
3.  Quantitative trait loci in hop (Humulus lupulus L.) reveal complex genetic architecture underlying variation in sex, yield and cone chemistry 
BMC Genomics  2013;14:360.
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.
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.
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.
PMCID: PMC3680207  PMID: 23718194
Linkage map; Transferable marker; Diversity arrays technology DArT; Pleiotropy; Sex-linked marker; Dry cone weight; Plant secondary metabolite; Hop acid; Essential oil
4.  A consensus map of rapeseed (Brassica napus L.) based on diversity array technology markers: applications in genetic dissection of qualitative and quantitative traits 
BMC Genomics  2013;14:277.
Dense consensus genetic maps based on high-throughput genotyping platforms are valuable for making genetic gains in Brassica napus through quantitative trait locus identification, efficient predictive molecular breeding, and map-based gene cloning. This report describes the construction of the first B. napus consensus map consisting of a 1,359 anchored array based genotyping platform; Diversity Arrays Technology (DArT), and non-DArT markers from six populations originating from Australia, Canada, China and Europe. We aligned the B. napus DArT sequences with genomic scaffolds from Brassica rapa and Brassica oleracea, and identified DArT loci that showed linkage with qualitative and quantitative loci associated with agronomic traits.
The integrated consensus map covered a total of 1,987.2 cM and represented all 19 chromosomes of the A and C genomes, with an average map density of one marker per 1.46 cM, corresponding to approximately 0.88 Mbp of the haploid genome. Through in silico physical mapping 2,457 out of 3,072 (80%) DArT clones were assigned to the genomic scaffolds of B. rapa (A genome) and B. oleracea (C genome). These were used to orientate the genetic consensus map with the chromosomal sequences. The DArT markers showed linkage with previously identified non-DArT markers associated with qualitative and quantitative trait loci for plant architecture, phenological components, seed and oil quality attributes, boron efficiency, sucrose transport, male sterility, and race-specific resistance to blackleg disease.
The DArT markers provide increased marker density across the B. napus genome. Most of the DArT markers represented on the current array were sequenced and aligned with the B. rapa and B. oleracea genomes, providing insight into the Brassica A and C genomes. This information can be utilised for comparative genomics and genomic evolution studies. In summary, this consensus map can be used to (i) integrate new generation markers such as SNP arrays and next generation sequencing data; (ii) anchor physical maps to facilitate assembly of B. napus genome sequences; and (iii) identify candidate genes underlying natural genetic variation for traits of interest.
PMCID: PMC3641989  PMID: 23617817
5.  DNA repair and crossing over favor similar chromosome regions as discovered in radiation hybrid of Triticum 
BMC Genomics  2012;13:339.
The uneven distribution of recombination across the length of chromosomes results in inaccurate estimates of genetic to physical distances. In wheat (Triticum aestivum L.) chromosome 3B, it has been estimated that 90% of the cross over events occur in distal sub-telomeric regions representing 40% of the chromosome. Radiation hybrid (RH) mapping which does not rely on recombination is a strategy to map genomes and has been widely employed in animal species and more recently in some plants. RH maps have been proposed to provide i) higher and ii) more uniform resolution than genetic maps, and iii) to be independent of the distribution patterns observed for meiotic recombination. An in vivo RH panel was generated for mapping chromosome 3B of wheat in an attempt to provide a complete scaffold for this ~1 Gb segment of the genome and compare the resolution to previous genetic maps.
A high density RH map with 541 marker loci anchored to chromosome 3B spanning a total distance of 1871.9 cR was generated. Detailed comparisons with a genetic map of similar quality confirmed that i) the overall resolution of the RH map was 10.5 fold higher and ii) six fold more uniform. A significant interaction (r = 0.879 at p = 0.01) was observed between the DNA repair mechanism and the distribution of crossing-over events. This observation could be explained by accepting the possibility that the DNA repair mechanism in somatic cells is affected by the chromatin state in a way similar to the effect that chromatin state has on recombination frequencies in gametic cells.
The RH data presented here support for the first time in vivo the hypothesis of non-casual interaction between recombination hot-spots and DNA repair. Further, two major hypotheses are presented on how chromatin compactness could affect the DNA repair mechanism. Since the initial RH application 37 years ago, we were able to show for the first time that the iii) third hypothesis of RH mapping might not be entirely correct.
PMCID: PMC3443642  PMID: 22827734
Non homologous end joining; Physical mapping; Gamma radiation; Deletion mutant; Chromatin; Wheat chromosome 3B; Radiation hybrid
6.  A reference linkage map for Eucalyptus 
BMC Genomics  2012;13:240.
Genetic linkage maps are invaluable resources in plant research. They provide a key tool for many genetic applications including: mapping quantitative trait loci (QTL); comparative mapping; identifying unlinked (i.e. independent) DNA markers for fingerprinting, population genetics and phylogenetics; assisting genome sequence assembly; relating physical and recombination distances along the genome and map-based cloning of genes. Eucalypts are the dominant tree species in most Australian ecosystems and of economic importance globally as plantation trees. The genome sequence of E. grandis has recently been released providing unprecedented opportunities for genetic and genomic research in the genus. A robust reference linkage map containing sequence-based molecular markers is needed to capitalise on this resource. Several high density linkage maps have recently been constructed for the main commercial forestry species in the genus (E. grandis, E. urophylla and E. globulus) using sequenced Diversity Arrays Technology (DArT) and microsatellite markers. To provide a single reference linkage map for eucalypts a composite map was produced through the integration of data from seven independent mapping experiments (1950 individuals) using a marker-merging method.
The composite map totalled 1107 cM and contained 4101 markers; comprising 3880 DArT, 213 microsatellite and eight candidate genes. Eighty-one DArT markers were mapped to two or more linkage groups, resulting in the 4101 markers being mapped to 4191 map positions. Approximately 13% of DArT markers mapped to identical map positions, thus the composite map contained 3634 unique loci at an average interval of 0.31 cM.
The composite map represents the most saturated linkage map yet produced in Eucalyptus. As the majority of DArT markers contained on the map have been sequenced, the map provides a direct link to the E. grandis genome sequence and will serve as an important reference for progressing eucalypt research.
PMCID: PMC3436727  PMID: 22702473
7.  Aluminum tolerance association mapping in triticale 
BMC Genomics  2012;13:67.
Crop production practices and industrialization processes result in increasing acidification of arable soils. At lower pH levels (below 5.0), aluminum (Al) remains in a cationic form that is toxic to plants, reducing growth and yield. The effect of aluminum on agronomic performance is particularly important in cereals like wheat, which has promoted the development of programs directed towards selection of tolerant forms. Even in intermediately tolerant cereals (i.e., triticale), the decrease in yield may be significant. In triticale, Al tolerance seems to be influenced by both wheat and rye genomes. However, little is known about the precise chromosomal location of tolerance-related genes, and whether wheat or rye genomes are crucial for the expression of that trait in the hybrid.
A mapping population consisting of 232 advanced breeding triticale forms was developed and phenotyped for Al tolerance using physiological tests. AFLP, SSR and DArT marker platforms were applied to obtain a sufficiently large set of molecular markers (over 3000). Associations between the markers and the trait were tested using General (GLM) and Multiple (MLM) Linear Models, as well as the Statistical Machine Learning (SML) approach. The chromosomal locations of candidate markers were verified based on known assignments of SSRs and DArTs or by using genetic maps of rye and triticale.
Two candidate markers on chromosome 3R and 9, 15 and 11 on chromosomes 4R, 6R and 7R, respectively, were identified. The r2 values were between 0.066 and 0.220 in most cases, indicating a good fit of the data, with better results obtained with the GML than the MLM approach. Several QTLs on rye chromosomes appeared to be involved in the phenotypic expression of the trait, suggesting that rye genome factors are predominantly responsible for Al tolerance in triticale.
The Diversity Arrays Technology was applied successfully to association mapping studies performed on triticale breeding forms. Statistical approaches allowed the identification of numerous markers associated with Al tolerance. Available rye and triticale genetic maps suggested the putative location of the markers and demonstrated that they formed several linked groups assigned to distinct chromosomes (3R, 4R, 6R and 7R). Markers associated with genomic regions under positive selection were identified and indirectly mapped in the vicinity of the Al-tolerant markers. The present findings were in agreement with prior reports.
PMCID: PMC3313894  PMID: 22330691
8.  DArT markers for the rye genome - genetic diversity and mapping 
BMC Genomics  2009;10:578.
Implementation of molecular breeding in rye (Secale cereale L.) improvement programs depends on the availability of high-density molecular linkage maps. However, the number of sequence-specific PCR-based markers available for the species is limited. Diversity Arrays Technology (DArT) is a microarray-based method allowing for detection of DNA polymorphism at several thousand loci in a single assay without relying on DNA sequence information. The objective of this study was the development and application of Diversity Arrays technology for rye.
Using the PstI/TaqI method of complexity reduction we created a rye diversity panel from DNA of 16 rye varieties and 15 rye inbred lines, including parents of a mapping population consisting of 82 recombinant inbred lines. The usefulness of a wheat diversity panel for identification of DArT markers for rye was also demonstrated. We identified 1022 clones that were polymorphic in the genotyped ILs and varieties and 1965 clones that differentiated the parental lines L318 and L9 and segregated in the mapping population. Hierarchical clustering and ordination analysis were performed based on the 1022 DArT markers to reveal genetic relationships between the rye varieties and inbred lines included in the study. Chromosomal location of 1872 DArT markers was determined using wheat-rye addition lines and 1818 DArT markers (among them 1181 unique, non-cosegregating) were placed on a genetic linkage map of the cross L318 × L9, providing an average density of one unique marker every 2.68 cM. This is the most saturated rye linkage map based solely on transferable markers available at the moment, providing rye breeders and researches with a better choice of markers and a higher probability of finding polymorphic markers in the region of interest.
The Diversity Arrays Technology can be efficiently and effectively used for rye genome analyses - assessment of genetic similarity and linkage mapping. The 11520-clone rye genotyping panel with several thousand markers with determined chromosomal location and accessible through an inexpensive genotyping service is a valuable resource for studies on rye genome organization and in molecular breeding of the species.
PMCID: PMC2795769  PMID: 19958552
9.  Development and mapping of DArT markers within the Festuca - Lolium complex 
BMC Genomics  2009;10:473.
Grasses are among the most important and widely cultivated plants on Earth. They provide high quality fodder for livestock, are used for turf and amenity purposes, and play a fundamental role in environment protection. Among cultivated grasses, species within the Festuca-Lolium complex predominate, especially in temperate regions. To facilitate high-throughput genome profiling and genetic mapping within the complex, we have developed a Diversity Arrays Technology (DArT) array for five grass species: F. pratensis, F. arundinacea, F. glaucescens, L. perenne and L. multiflorum.
The DArTFest array contains 7680 probes derived from methyl-filtered genomic representations. In a first marker discovery experiment performed on 40 genotypes from each species (with the exception of F. glaucescens for which only 7 genotypes were used), we identified 3884 polymorphic markers. The number of DArT markers identified in every single genotype varied from 821 to 1852. To test the usefulness of DArTFest array for physical mapping, DArT markers were assigned to each of the seven chromosomes of F. pratensis using single chromosome substitution lines while recombinants of F. pratensis chromosome 3 were used to allocate the markers to seven chromosome bins.
The resources developed in this project will facilitate the development of genetic maps in Festuca and Lolium, the analysis on genetic diversity, and the monitoring of the genomic constitution of the Festuca × Lolium hybrids. They will also enable marker-assisted selection for multiple traits or for specific genome regions.
PMCID: PMC2770082  PMID: 19832973
10.  DArT markers: diversity analyses, genomes comparison, mapping and integration with SSR markers in Triticum monococcum 
BMC Genomics  2009;10:458.
Triticum monococcum (2n = 2x = 14) is an ancient diploid wheat with many useful traits and is used as a model for wheat gene discovery. DArT (Diversity Arrays Technology) employs a hybridisation-based approach to type thousands of genomic loci in parallel. DArT markers were developed for T. monococcum to assess genetic diversity, compare relationships with hexaploid genomes, and construct a genetic linkage map integrating DArT and microsatellite markers.
A DArT array, consisting of 2304 hexaploid wheat, 1536 tetraploid wheat, 1536 T. monococcum as well as 1536 T. boeoticum representative genomic clones, was used to fingerprint 16 T. monococcum accessions of diverse geographical origins. In total, 846 polymorphic DArT markers were identified, of which 317 were of T. monococcum origin, 246 of hexaploid, 157 of tetraploid, and 126 of T. boeoticum genomes. The fingerprinting data indicated that the geographic origin of T. monococcum accessions was partially correlated with their genetic variation. DArT markers could also well distinguish the genetic differences amongst a panel of 23 hexaploid wheat and nine T. monococcum genomes. For the first time, 274 DArT markers were integrated with 82 simple sequence repeat (SSR) and two morphological trait loci in a genetic map spanning 1062.72 cM in T. monococcum. Six chromosomes were represented by single linkage groups, and chromosome 4Am was formed by three linkage groups. The DArT and SSR genetic loci tended to form independent clusters along the chromosomes. Segregation distortion was observed for one third of the DArT loci. The Ba (black awn) locus was refined to a 23.2 cM region between the DArT marker locus wPt-2584 and the microsatellite locus Xgwmd33 on 1Am; and the Hl (hairy leaf) locus to a 4.0 cM region between DArT loci 376589 and 469591 on 5Am.
DArT is a rapid and efficient approach to develop many new molecular markers for genetic studies in T. monococcum. The constructed genetic linkage map will facilitate localisation and map-based cloning of genes of interest, comparative mapping as well as genome organisation and evolution studies between this ancient diploid species and other crops.
PMCID: PMC2764732  PMID: 19788762
11.  New DArT markers for oat provide enhanced map coverage and global germplasm characterization 
BMC Genomics  2009;10:39.
Genomic discovery in oat and its application to oat improvement have been hindered by a lack of genetic markers common to different genetic maps, and by the difficulty of conducting whole-genome analysis using high-throughput markers. This study was intended to develop, characterize, and apply a large set of oat genetic markers based on Diversity Array Technology (DArT).
Approximately 19,000 genomic clones were isolated from complexity-reduced genomic representations of pooled DNA samples from 60 oat varieties of global origin. These were screened on three discovery arrays, with more than 2000 polymorphic markers being identified for use in this study, and approximately 2700 potentially polymorphic markers being identified for use in future studies. DNA sequence was obtained for 2573 clones and assembled into a non-redundant set of 1770 contigs and singletons. Of these, 705 showed highly significant (Expectation < 10E-10) BLAST similarity to gene sequences in public databases. Based on marker scores in 80 recombinant inbred lines, 1010 new DArT markers were used to saturate and improve the 'Kanota' × 'Ogle' genetic map. DArT markers provided map coverage approximately equivalent to existing markers. After binning markers from similar clones, as well as those with 99% scoring similarity, a set of 1295 non-redundant markers was used to analyze genetic diversity in 182 accessions of cultivated oat of worldwide origin. Results of this analysis confirmed that major clusters of oat diversity are related to spring vs. winter type, and to the presence of major breeding programs within geographical regions. Secondary clusters revealed groups that were often related to known pedigree structure.
These markers will provide a solid basis for future efforts in genomic discovery, comparative mapping, and the generation of an oat consensus map. They will also provide new opportunities for directed breeding of superior oat varieties, and guidance in the maintenance of oat genetic diversity.
PMCID: PMC2661094  PMID: 19159465
12.  A MITE-based genotyping method to reveal hundreds of DNA polymorphisms in an animal genome after a few generations of artificial selection 
BMC Genomics  2008;9:459.
For most organisms, developing hundreds of genetic markers spanning the whole genome still requires excessive if not unrealistic efforts. In this context, there is an obvious need for methodologies allowing the low-cost, fast and high-throughput genotyping of virtually any species, such as the Diversity Arrays Technology (DArT). One of the crucial steps of the DArT technique is the genome complexity reduction, which allows obtaining a genomic representation characteristic of the studied DNA sample and necessary for subsequent genotyping. In this article, using the mosquito Aedes aegypti as a study model, we describe a new genome complexity reduction method taking advantage of the abundance of miniature inverted repeat transposable elements (MITEs) in the genome of this species.
Ae. aegypti genomic representations were produced following a two-step procedure: (1) restriction digestion of the genomic DNA and simultaneous ligation of a specific adaptor to compatible ends, and (2) amplification of restriction fragments containing a particular MITE element called Pony using two primers, one annealing to the adaptor sequence and one annealing to a conserved sequence motif of the Pony element. Using this protocol, we constructed a library comprising more than 6,000 DArT clones, of which at least 5.70% were highly reliable polymorphic markers for two closely related mosquito strains separated by only a few generations of artificial selection. Within this dataset, linkage disequilibrium was low, and marker redundancy was evaluated at 2.86% only. Most of the detected genetic variability was observed between the two studied mosquito strains, but individuals of the same strain could still be clearly distinguished.
The new complexity reduction method was particularly efficient to reveal genetic polymorphisms in Ae. egypti. Overall, our results testify of the flexibility of the DArT genotyping technique and open new prospects as regards its application to a wider range of species, including animals which have been refractory to it so far. DArT has also a role to play in the current burst of whole-genome scans carried out in various organisms, which track signatures of selection in order to unravel the basis of genetic adaptation.
PMCID: PMC2579443  PMID: 18837997
13.  DArT markers: diversity analyses and mapping in Sorghum bicolor 
BMC Genomics  2008;9:26.
The sequential nature of gel-based marker systems entails low throughput and high costs per assay. Commonly used marker systems such as SSR and SNP are also dependent on sequence information. These limitations result in high cost per data point and significantly limit the capacity of breeding programs to obtain sufficient return on investment to justify the routine use of marker-assisted breeding for many traits and particularly quantitative traits. Diversity Arrays Technology (DArT™) is a cost effective hybridisation-based marker technology that offers a high multiplexing level while being independent of sequence information. This technology offers sorghum breeding programs an alternative approach to whole-genome profiling. We report on the development, application, mapping and utility of DArT™ markers for sorghum germplasm.
A genotyping array was developed representing approximately 12,000 genomic clones using PstI+BanII complexity with a subset of clones obtained through the suppression subtractive hybridisation (SSH) method. The genotyping array was used to analyse a diverse set of sorghum genotypes and screening a Recombinant Inbred Lines (RIL) mapping population. Over 500 markers detected variation among 90 accessions used in a diversity analysis. Cluster analysis discriminated well between all 90 genotypes. To confirm that the sorghum DArT markers behave in a Mendelian manner, we constructed a genetic linkage map for a cross between R931945-2-2 and IS 8525 integrating DArT and other marker types. In total, 596 markers could be placed on the integrated linkage map, which spanned 1431.6 cM. The genetic linkage map had an average marker density of 1/2.39 cM, with an average DArT marker density of 1/3.9 cM.
We have successfully developed DArT markers for Sorghum bicolor and have demonstrated that DArT provides high quality markers that can be used for diversity analyses and to construct medium-density genetic linkage maps. The high number of DArT markers generated in a single assay not only provides a precise estimate of genetic relationships among genotypes, but also their even distribution over the genome offers real advantages for a range of molecular breeding and genomics applications.
PMCID: PMC2270266  PMID: 18208620
14.  A DArT platform for quantitative bulked segregant analysis 
BMC Genomics  2007;8:196.
Bulked segregant analysis (BSA) identifies molecular markers associated with a phenotype by screening two DNA pools of phenotypically distinct plants for markers with skewed allele frequencies. In contrast to gel-based markers, hybridization-based markers such as SFP, DArT or SNP generate quantitative allele-frequency estimates. Only DArT, however, combines this advantage with low development and assay costs and the ability to be deployed for any plant species irrespective of its ploidy level. Here we investigate the suitability of DArT for BSA applications using a barley array as an example.
In a first test experiment, we compared two bulks of 40 Steptoe/Morex DH plants with contrasting pubescent leaves (mPub) alleles on chromosome 3H. At optimized levels of experimental replication and marker-selection threshold, the BSA scan identified 433 polymorphic markers. The relative hybridization contrast between bulks accurately reflected the between-bulk difference in the frequency of the mPub allele (r = 0.96). The 'platform noise' of DArT assays, estimated by comparing two identical aliquots of a DNA mixture, was significantly lower than the 'pooling noise' reflecting the binomial sampling variance of the bulking process. The allele-frequency difference on chromosome 3H increased in the vicinity of mPub and peaked at the marker with the smallest distance from mPub (4.6 cM). In a validation experiment with only 20 plants per bulk we identified an aluminum (Al) tolerance locus in a Dayton/Zhepi2 DH population on chromosome 4H with < 0.8 cM precision, the same Al-tolerance locus that had been mapped before in other barley populations.
DArT-BSA identifies genetic loci that influence phenotypic characters in barley with at least 5 cM accuracy and should prove useful as a generic tool for high-throughput, quantitative BSA in plants irrespective of their ploidy level.
PMCID: PMC1920522  PMID: 17597543
15.  A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits 
BMC Genomics  2006;7:206.
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.
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.
PMCID: PMC1564146  PMID: 16904008

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