Isolated chromosomes as a new and efficient source of DArT markers ...

Theor Appl Genet (2010) 121:465–474 DOI 10.1007/s00122-010-1323-8

ORIGINAL PAPER

Isolated chromosomes as a new and efficient source of DArT markers for the saturation of genetic maps Peter Wenzl • Pavla Sucha´nkova´ • Jason Carling • Hana Sˇimkova´ Eric Huttner • Marie Kubala´kova´ • Pierre Sourdille • Edie Paul • Catherine Feuillet • Andrzej Kilian • Jaroslav Dolezˇel

•

Received: 21 December 2009 / Accepted: 5 March 2010 / Published online: 4 April 2010 Ó Springer-Verlag 2010

Abstract 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,

Communicated by M. Sorrells.

Electronic supplementary material The online version of this article (doi:10.1007/s00122-010-1323-8) contains supplementary material, which is available to authorized users. P. Wenzl  J. Carling  E. Huttner  A. Kilian Diversity Arrays Technology Pty Ltd, 1 Wilf Crane Crescent, Yarralumla, ACT 2600, Australia P. Wenzl International Maize and Wheat Improvement Center (CIMMYT), Apdo. Postal 6-641, 06600 Mexico DF, Mexico P. Sucha´nkova´  H. Sˇimkova´  M. Kubala´kova´  J. Dolezˇel (&) Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Sokolovska´ 6, 772 00 Olomouc, Czech Republic e-mail: [email protected] P. Sourdille  C. Feuillet INRA-UBP, UMR1095, Genetics Diversity and Ecophysiology of Cereals, Clermont-Ferrand, France E. Paul Geneflow Inc., PO Box 230748, Centreville, VA 20120, USA

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.

Introduction During the past two decades, the development of an impressive range of DNA marker types (Doveri et al. 2008; Nguyen and Wu 2005) enabled significant advances in studying genetic diversity, following the transmission of loci of interest and facilitating marker-assisted selection and positional gene cloning. In the context of building physical maps, DNA markers are also essential for ordering BAC contigs and anchoring them to genetic maps (Meyers et al. 2004), an application that requires high marker densities and well-saturated genetic maps. A large number of markers are especially important for crops with large genomes, such as barley, rye, and wheat. The hexaploid genome of bread wheat (Triticum aestivum L.)

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is approximately 17-Gbp-long, and consists of three subgenomes: A, B, and D, each comprising seven chromosomes (Bennett and Smith 1976). When building the first physical map of a wheat chromosome, Paux et al. (2008) used 1,443 markers to anchor 680 contigs representing 75% of the chromosome 3B physical map. If this proportion holds for every chromosome, a minimum of 25,000 markers would be needed for a similar coverage of the entire wheat genome. Paux et al. (2008) also demonstrated that different classes of markers exhibited different distributions along chromosome 3B. Consequently, a large number of different types of markers are needed to ensure optimal coverage of entire genomes. While markers are usually developed for entire genomes, an alternative option is to focus marker development on subgenomic regions. This approach should make it possible to generate high marker densities in regions of interest, such as a chromosome or a chromosome arm from which a gene is being cloned or for which a physical map is being constructed. Flow cytometric sorting and microdissection are the only methods suitable for isolation of DNA from selected chromosomes and chromosome arms (Dolezˇel et al. 2004). However, the throughput of microdissection is low, and only flow cytometry can sort large quantities of chromosomes. To date, protocols for chromosome-sorting are available for 17 plant species, including some important legumes and cereals (Dolezˇel et al. 2004). In bread wheat, flow cytometric chromosome analysis and sorting was reported by Vra´na et al. (2000). The resulting distribution of chromosome DNA content (flow karyotypes) comprised four peaks. While the largest wheat chromosome (3B) formed a discrete peak and was readily sortable, the remaining 20 chromosomes clustered into three peaks. Subsequently, Kubala´kova´ et al. (2002) demonstrated that with the exception of 3BL and 5BL, all wheat chromosome arms can be sorted as telosomes, which are maintained in stable cytogenetic stocks (Sears 1954). The 3BL and 5BL arms can be isolated from stocks carrying them as isochromosomes. A number of applications of flow-sorted chromosomes have penetrated plant genetics and genomics during the last decade. These included physical mapping using PCR and FISH (Vla´cˇilova´ et al. 2002; Vala´rik et al. 2004), the construction of chromosome and chromosome armspecific BAC libraries (Sˇafa´rˇ et al. 2004; Janda et al. 2004, 2006; Sˇimkova´ et al. 2008a), the mapping of SNPs using oligonucleotide arrays (Sˇimkova´ et al. 2008b), and shotgun-sequencing with the next generation sequencing technologies (Mayer et al. 2009). However, until now, DNA isolated from flow-sorted chromosomes has only been employed to develop SSR markers, and has not yet been combined with genotyping platforms with high multiplexing

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levels (Kofler et al. 2008; Roma´n et al. 2004; Pozˇa´rkova´ et al. 2002). Diversity arrays technology (DArT) markers are a relatively recent type of high-throughput marker system that excels in throughput and cost-effectiveness (Jaccoud et al. 2001; Wenzl et al. 2004; http://www.diversityarrays.com). DArT in its current implementation is a hybridizationbased genotyping method that does not require sequence information and uses microarray technology to identify and type several thousands of dominant markers in parallel (Kilian et al. 2005). The technology has been established for more than 50 species and relies on DNA polymorphisms at (SNPs, InDels, methylation differences) or between (InDels) restriction-enzyme sites. DArT markers are widely used to analyze genetic diversity and develop genetic maps (Wenzl et al. 2004; White et al. 2008). Semagn et al. (2006) showed that DArT markers were distributed differently across the genome than SSR and AFLP markers, while Wenzl et al. (2004, 2006) and Akbari et al. (2006) reported preferential locations of DArT markers in gene-rich, subtelomeric regions in barley and wheat, respectively. This study explores the possibility of developing DArT markers from specific regions of a plant genome by using DNA from flow-sorted chromosomes. We test this approach using the longest chromosome (3B) and one of the shortest chromosome arms (1BS) of wheat as pilots. We report not only on marker development but also on the saturation of the genetic map of chromosome 3B.

Materials and methods Plant material Seeds of five hexaploid wheat (Triticum aestivum L., 2n = 6x = 42) cultivars ‘Chinese Spring’, ‘Courtot’, ‘Renan’, and ‘Opata’, and synthetic wheat W7984 (Van Deynze et al. 1995) were obtained from INRA (ClermontFerrand, France). These cultivars were chosen because they were used as parents of international reference mapping populations (Opata 9 W7984: ITMI population, Van Deynze et al. 1995; Chinese Spring 9 Renan: IWGSC population; Chinese Spring 9 Courtot population, Sourdille et al. 2003). Two ditelosomic lines of hexaploid wheat cv. ‘Pavon 76’ (2n = 40 ? 2t1BS), carrying telocentric chromosomes 1BS of cultivars ‘Begra’ and ‘Henika’, were developed by centric misdivision of single chromosome substitution lines 1B of these cultivars into ‘Pavon’ by Prof. Adam Lukaszewski (University of California, Riverside, USA). The seeds were germinated in the dark at 25 ± 0.5°C on moistened filter paper for 3 days to obtain a root length of approximately 2–3 cm.

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Chromosome sorting Mitotic metaphase chromosomes were sorted using flow cytometry according to Vra´na et al. (2000). To prepare liquid suspensions of intact chromosomes, roots of young seedlings were sequentially treated with hydroxyurea and amiprophos-methyl to accumulate meristematic root-tip cells at metaphase, and the synchronized root-tip meristems were fixed with formaldehyde (Vra´na et al. 2000). Chromosomes were released by mechanical homogenization of 25 root tips in 1 mL ice-cold LB01 buffer (Dolezˇel et al. 1989) and stained by 2 lg mL-1 DAPI (40 ,6-diamidino-2phenylindole). The samples were processed using a FACSVantage SE flow cytometer and sorter (Becton Dickinson, San Jose´, USA). Batches of 10,000 chromosomes 3B and 35,000 telosomes 1BS were sorted into 50 lL deionized water in a PCR tube. The purity of sorted fractions was evaluated by FISH using probes for GAA microsatellite, Afa and telomeric repeat as described in Kubala´kova´ et al. (2002). DNA extraction The DNA was prepared from flow-sorted chromosomes according to Sˇimkova´ et al. (2008b). Briefly, the chromosomes were treated with proteinase K in a buffer consisting of 2.5 mM Tris (pH 8.0), 1.25 mM EDTA (pH 8.0), and 0.125% (w/v) SDS under gentle shaking at 50°C for 40 h. Two and 3 lg of proteinase K were added to the 10,000 and 35,000 chromosome samples, respectively. Another 1 or 1.5 lg was added after 20 h of treatment. The proteinase K and the buffer were subsequently removed using a Microcon YM-100 column (Millipore Corporation, Bedford, USA) in four rounds of centrifugation at 5009g for 14 min at 23°C. The amount of purified DNA was estimated using fluorometry. Preparing DArT arrays containing chromosomespecific markers Genomic representations were generated by digesting 1–2 ng of flow-sorted 1BS or 3B DNA with two units of both PstI and TaqI (NEB, Beverly, USA). A PstI adapter (50 -AC GAT GGA TCC AGT GCA-30 annealed with 50 -CTG GAT CCA TCG TGC A-30 ) was simultaneously ligated to PstI ends using T4 DNA ligase (NEB). Approximately 5% of the ligation product was amplified in a 25-lL reaction using 50 -AT GGA TCC AGT GCA G-30 as a primer. The amplification program was 94°C for 1 min, followed by 30 cycles of 94°C for 20 s, 58°C for 40 s, 72°C for 1 min, and 72°C for 7 min. Fragments from the resulting genomic representations were cloned into the pCR2.1-TOPO vector

467

(Invitrogen, Mount Waverley, Australia) (Wenzl et al. 2004; Akbari et al. 2006). Individual clones (384 clones derived from the two ditelosomic 1BS lines, 1,536 clones derived from chromosome 3B of ‘Chinese Spring’ and 1,152 3B-derived clones from the mixture of four other wheat cultivars; see Table 1) were grown in 384-well plates containing freezing medium consisting of 0.7 9 LB medium supplemented with 4.4% glycerol, 8.21 g L-1 K2HPO4, 1.80 g L-1 KH2PO4, 0.50 g L-1 Na3-citrate, 0.10 g L-1 MgSO47H2O and 0.90 g L-1 (NH4)2SO4, 100 mg L-1 ampicillin, and 100 mg L-1 kanamycin. Inserts were amplified from 0.5 lL aliquots of bacterial cultures in 25 lL reactions containing 50 mM Tris, 6 mM HCl, 16 mM (NH4)2SO4, 1.5 mM MgCl2, 200 lM dNTPs, 0.2 lM M13f primer (50 -GTT TTC CCA GTC ACG ACG TTG-30 ), 0.2 lM M13r primer (50 -TGA GCG GAT AAC AAT TTC ACA CAG -30 ), and a sufficient amount of Taq polymerase to obtain close-to-maximum yield. Amplification conditions were 95°C for 4 min, 57°C for 35 s, 72°C for 1 min, followed by 35 cycles of 94°C for 35 s, 52°C for 35 s, 72°C for 1 min, and 72°C for 7 min. Amplification reactions were dried at 37°C for 24 h, washed with 77% ethanol, dried at 37°C for 1 h, and dissolved in DArTspotter buffer (50% DMSO, 1.5 M sorbitol, 0.1 M tri-ethanolamine–HCl, 0.5% (w/v) dextran, 0.02% (w/v) CHAPS). The amplified inserts were grouped with other DArT markers derived from 234 whole-genome DNA samples of bread wheat, durum wheat and diploid, tetraploid and hexaploid wheat relatives, and printed in duplicate spots on SuperChip poly-L-lysine slides (Erie Microarray, Portsmouth, NH, USA) using a MicroGrid II arrayer (Biorobotics, Cambridge, UK). At least 24 h after printing,

Table 1 Purity of the chromosome fractions sorted from hexaploid wheat Chromosome

Cultivar

Purity (%)a Mean

SD

3B

Chinese Spring

88

1.58

3B

Courtot

92

2.08

3B

Opata

90

2.51

3B

Renan

93

1.53

3B

Synthetic

91

1.00

1BS

Begra

84

1.15

1BS

Henika

87

3.79

a

Estimated by FISH independently on three microscope slides per cultivar. On each slide, 100 sorted particles were evaluated. The chromosomes were identified with probes for GAA microsatellite, Afa repeat family, and telomeric repeat

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the slides were immersed in 92°C hot water for 2 min, transferred to a solution containing 100 lM DTT and 100 lM EDTA, and dried by centrifugation at 5009g for 7 min followed by incubation under vacuum for 30 min (Wenzl et al. 2004; Akbari et al. 2006). DArT genotyping procedure Approximately 20–100 ng of DNA samples of individual plants belonging to 19 different wheat mapping populations (with between 90 and 200 plants per mapping population) were processed as described above. Two replicate genomic representations were amplified from each sample, pooled, precipitated with one volume of iso-propanol, centrifuged at [3,0009g for 40 min at 30°C, washed with 77% ethanol, centrifuged at [3,0009g for 40 min at 30°C, and dried at room temperature. The representations were then labeled by adding 5 lL of labeling mix [10 mM Tris– Cl (pH 7.9), 10 mM MgC12, 50 mM NaC1, 1 mM DTT, 50 lM random decamers, 0.2 mM of dATP, dCTP, and dGTP, and 20 lM of dTTP], denaturing the solution at 95°C for 3 min, adding 5 lL of dye mix [10 mM Tris–Cl (pH 7.9), 10 mM MgC12, 50 mM NaC1, 1 mM DTT, 20 lM of either Cy3-dUTP or Cy5-dUTP (GE Healthcare, Rydalmere, Australia)], and 20 U lL-1 Klenow exo(NEB), and incubating the mixture at 37°C for 3 h (Wenzl et al. 2004; Akbari et al. 2006). The resulting targets were added to 50 lL of a 500:50:2.5:2 mixture of ExpressHyb solution (Clonetech, Cheltenham, Australia), 10 mg mL-1 denatured herringsperm DNA (Promega, Alexandria, Australia), 0.7 mg mL-1 FAM-labeled pCR2.1-TOPO polylinker (amplified with M13f and M13r primers), and 0.5 M EDTA (pH 8.0). The mixtures were denatured at 95°C for 3 min and hybridized to DArT arrays in a water bath at 62°C for 16 h. After hybridization, slides were washed in 19 SSC, 0.1% SDS (5 min), 19 SSC (5 min), 0.29 SSC (1 min), 0.029 SSC (30 s), and 100 lM DTT (30 s), and dried by centrifugation at 5009g for 7 min followed by incubation under vacuum for 30 min. The slides were scanned with a Tecan LS300 confocal laser scanner at 488, 543, and 633 nm (Tecan Austria, Gro¨dig, Austria). Slide images were analyzed with DArTsoft (Diversity Arrays Technology P/L, Canberra, Australia) to identify polymorphic markers and call their allelic states (Wenzl et al. 2004; Akbari et al. 2006). Constructing a consensus map for chromosome 3B A total of 19 DH, RIL, and F2 wheat populations were genotyped for this study. All populations were genotyped with a set of whole genome-derived DArT markers which expanded from 3,456 to 6,528 over the period of

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approximately 2.5 years (polymorphic 1BS-derived markers were also added during this process). Four of these 19 populations were also genotyped with the 2,688 chromosome 3B-derived DArT markers, which were more recently generated using methods described above. The F2 population originates from a Chinese Spring 9 Renan cross, and is described in detail in Paux et al. (2008). The other maps were derived from DH and RIL populations genotyped at Triticarte P/L, with permission of the population owners to utilize the segregation data for mapping purposes. It is anticipated that the identity of all parents of these populations and possibly the segregation data as well will become available when consensus maps of all wheat chromosomes are generated and published. Markers mapping to chromosome 3B were identified through linkage with other DArT markers for which chromosome assignments had previously been established. The marker order in each of the 20 chromosome-3B data sets (the F2 population data were split into a maternal and a paternal dataset) was established with EasyMap (Wenzl et al., in preparation), a program that implements the RECORD marker-ordering algorithm described by van Os et al. (2005). Poorly fitting markers (with more than 3% apparent double crossovers) were removed, and the marker order was re-optimized. Potential genotyping errors (LODerror [ 4; Lincoln and Lander 1992) were substituted with missing data, and the marker order was re-optimized again. The individual maps were then oriented and fed into PhenoMap software to construct a synthetic consensus map (GeneFlow Inc., Alexandria, USA). PhenoMap used the map with the largest number of markers to establish the order for all bridging markers it contains. It then added the remaining bridging markers in an iterative fashion by processing the remaining maps in descending order of the number of markers they have in common with the growing consensus map. Once all common markers were ordered, unique markers were added to the resulting map. To place a unique marker, its relative distance to the nearest flanking common markers on the component map was calculated and scaled to the equivalent distance on the consensus map.

Results Chromosome sorting and preparation of DNA Flow cytometric analysis of DAPI-stained chromosomes isolated from hexaploid wheat cv. ‘Chinese Spring’ results in histograms of relative fluorescence intensity (flow karyotypes) with four clearly resolved peaks (Fig. 1a). Three composite peaks (I, II, and III) contain various groups of chromosomes, while the rightmost peak is made of chromosome 3B, the largest wheat chromosome (Kubala´kova´ et al. 2002).

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469

A

B

III

1BS (314Mbp)

III

I II

I II 3B (993Mbp)

Fig. 1 Histograms of relative fluorescence intensities (flow karyotypes) obtained during the analysis of DAPI-stained mitotic chromosomes of bread wheat (T. aestivum). a Flow karyotype of cv. ‘Chinese Spring’ (2n = 6x = 42) consists of three composite peaks (I–III) representing groups of chromosomes and a peak representing chromosome 3B. Chromosomes were sorted from the 3B peak for DNA isolation. b Flow karyotype of a ditelosomic line of cv. ‘Pavon

76’ (2n = 40 ? 2t1BS) carrying a telocentric chromosome 1BS of cv. ‘Henika’, which was sorted from the well-resolved peak. Molecular sizes of 3B and 1BS were determined considering 1C genome size 16,937 Mbp (Bennett and Smith 1976) and relative chromosome lengths as given by Gill et al. (1991). X-axis relative DAPI fluorescence intensity; Y-axis number of events

Similar flow karyotypes were observed after analyzing chromosome suspensions prepared from cultivars ‘Courtot’, ‘Renan’, ‘W7984’ and ‘Opata’ (data not shown) that were used for preparing the chromosome-specific DArT arrays in this study. The analysis of ditelosomic lines of cv. ‘Pavon 76’ carrying short arms of chromosome 1B (1BS) of cvs. ‘Begra’ and ‘Henika’ produced flow karyotypes that contained all four regular peaks (I–III, 3B) and an additional peak on the left of the composite peak I, representing chromosome 1BS (Fig. 1b). Chromosome 3B and chromosome arm 1BS were sorted in batches of 10,000 and 35,000 copies, respectively. Sorting was performed at rates of 4–6 s-1 for 3B and 20 s-1 for 1BS, and one batch of chromosomes took approximately 45 min to sort. The average purity in the sorted fractions ranged from 88 to 93% for 3B and from 84 to 87% for 1BS, as determined by FISH on chromosomes sorted onto a microscopic slide using probes for GAA microsatellite, Afa and telomeric repeat (Table 1). The amounts of DNA obtained from individual batches of sorted chromosomes ranged from 6 to 15 ng per batch. This quantity was adequate as less than 5 ng of DNA is sufficient to prepare the genomic representations for the development of DArT markers.

populations (Akbari et al. 2006; Triticarte P/L, unpublished data) (Fig. 2). Fifty-nine of the 68 polymorphic DArT markers (86.7%) derived from the 384 chromosome-1BS clones were assigned to chromosome 1B. Two of them were also mapped to a second locus on a different chromosome (5B and 7B). Another two markers (2.9%) showed linkage to homoeologous chromosomes 1A and 1D. Among the 711 polymorphic DArT markers derived from the 2,688 chromosome-3B clones, 553 (77.8%) were assigned to chromosome 3B. Four of them were multi-locus markers that reported a second locus on one of the two homoeologous chromosomes (3A, 3D). Twenty-five of the 711 polymorphic markers (3.5%) were exclusively assigned to homoeologous chromosomes 3A and 3D but not 3B.

Efficiency of chromosome-specific DArT marker development To estimate the efficiency of DArT marker development from a specific chromosome (or chromosome arm), we identified genetic linkage between the 1BS- or 3B-derived markers and a set of several thousand DArT markers whose chromosomal locations had previously been established by analyzing the segregation data of approximately 100 wheat

A consensus map for chromosome 3B Having confirmed the effectiveness of targeting marker discovery to specific chromosomes by using flow-sorted chromosomes, we next explored the degree of map saturation that could be achieved with this approach. We focused on chromosome 3B because of the greater number of markers developed for this chromosome. A consensus map of hexaploid wheat was built using the PhenoMap software to merge the order of markers present in 20 individual maps derived from 19 bi-parental populations (the F2-population dataset was split into two separate parental maps). The consensus map contains 939 markers, including 269 whole genome-derived DArT markers, 510 chromosome-3B-derived DArT markers, and 160 other markers (mostly SSR) (Fig. 3; ESM1). One whole genomederived DArT marker (wPt-8718) was mapped to two separate loci within chromosome 3B, thus bringing the total number of loci on the consensus genetic map to 940.

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A

70

1B other

No. of markers

60 50 40 30 20 5 of 9 markers

10 0

B

700

3B other

No. of markers

600 500 400 300 200

68 of 158 markers

100 0

A

B

D

1

Sub-genome

2

3

4

5

6

7

Homeologous group

Fig. 2 Efficiency of targeting marker discovery to selected chromosomes. DArT markers cloned from genomic representations of flowsorted 1BS chromosome arms (a) and 3B chromosomes (b) were mapped to chromosomes by comparing their segregation patterns in bi-parental mapping populations against those of a set of whole genome-derived DArT markers for which chromosome assignments

had been established previously. Black bars denote markers assigned to the chromosomes from which markers had been derived. Gray bars represent markers assigned to other chromosomes. The small inserts in the two left charts indicate the number of markers mapping to nontarget chromosomes on the B genome as a fraction to the total number of markers mapping to any of the non-target chromosomes

It is noteworthy that only 2,688 3B-derived clones (probes) needed to be screened to obtain almost twice as many polymorphic 3B markers (510) as identified previously by screening about 70,000 whole genome-derived clones (269). Approximately one-third of the 940 loci on the consensus map (304) were genetically distinct ([0 cM distance between adjacent locus pairs at the resolution level of this study). DArT markers were mapped to 237 of these genetically distinct loci (77.7%) which was in accordance with their representativeness among the whole set of loci (83%). SSR markers mapped to 101 of these loci (33.4%). DArT markers cloned from whole-genome DNA preparations covered 142 loci, while those derived from flowsorted 3B chromosomes covered 132 loci (Table 2). To quantify more accurately the map-saturation improvement achieved by targeting marker discovery to flow-sorted 3B chromosomes, we removed from the consensus map all SSR markers and retained only those whole genome-derived DArT markers, which were polymorphic in the four populations used to map the 3B-derived markers. The number of genetically distinct loci more than doubled (from 92 to 192) when the 3B dataset is added to the whole-genome dataset (Table 2), despite the very limited genepool from which the 3B-specific markers had been developed (Table 1). This indicates that there is a

great potential to further saturate the 3B consensus map by sorting chromosomes from a larger number of genetically diverse accessions.

123

Discussion This study has explored the possibility of saturating subgenomic regions of genetic maps of plant genomes by generating DArT markers from specific chromosomes and chromosome arms. The DNA which is needed to isolate such markers can be prepared from flow-sorting isolated chromosomes, a technique that has been applied to 17 plant species to this date (Dolezˇel et al. 2004). We chose wheat with its complex, hexaploid genome to test this approach. Within wheat, we selected chromosomes with contrasting molecular sizes as test cases for this pilot study: chromosome 3B (993 Mbp), the longest chromosome of wheat, and chromosome arm 1BS, which is among the shortest chromosome arms with 314 Mbp (Dolezˇel et al. 2009). Chromosome sorting and preparation of DNA Flow cytometric analysis of chromosomes stained by DAPI resulted in flow karyotypes, which corresponded to those obtained in earlier studies (Kubala´kova´ et al. 2002).

cfb6016 cfp78 cs-ssr20 gwm493

24.2 24.6

50.0

49.9

49.8

49.7

49.4

49.2

48.2 49.0 49.1

47.4

47.0 47.1

46.8

41.1 41.4 41.9 42.4 43.8 44.5 46.3 46.5 46.7

40.8

39.0 39.1 39.2 39.7 40.1 40.5 40.6

38.2

30.9 31.7 32.2 32.5 33.7 35.9 36.3 36.8 36.9

30.6

28.1 28.3 28.4 28.7 28.9 29.3 29.5 29.9

28.0

27.9

25.4 26.0 26.6 27.3 27.6

wPt-3231 wPt-4363 wPt-5916 wm1 wPt-7212 wPt-10945 wPt-11478 wPt-5399 wPt-0267 wPt-10647 tPt-6487 wPt-0571 wPt-10252 wPt-4653 wPt-4842 wPt-7132 wPt-9010 cfp57 cfp58 cfp59 wmc754 wPt-10018 wPt-11269 wPt-11483 wPt-1682 wPt-5064 wPt-7271 wPt-10991 wPt-5861 wmm1831 wPt-10585 wPt-8692 cfp45 nw2711 cfp171 cfd79 wPt-4849 wPt-3548 fbb185 wPt-10111 wPt-10354 wPt-10796 wPt-5750 wPt-8400 barc131 wPt-8303 wPt-10254 wPt-10473 wPt-10557 wPt-10926 wPt-11145 wPt-3878 cfb3119 wPt-3431 wPt-11261 wPt-3038 wmm1344 stm543tcac wPt-10024 wPt-3713 wPt-3797 wPt-0610 wPt-0250 wPt-10436 wPt-10611 wPt-5432 wPt-5442 wPt-8238 wPt-9041 wPt-0086 wPt-4739 wPt-8910 wmc623 cfb3023 nw216 cfp1133 cfp1260 cfp1274 gpw3092 wmm1441 wPt-0013 wPt-0895 wPt-1776 wPt-2193 wPt-10256 wPt-10761 wPt-2299 wPt-5318 wPt-11446 wPt-1691 wPt-2298 wPt-2766 wPt-5901 wPt-7409 wPt-9801 wPt-0773 wPt-10051 wPt-0688 wPt-10847 wPt-5836 wPt-6906 cfp127 cdo395 wmc78 wPt-6802 wmc43 wPt-8356 wPt-10954 wPt-9545 wPt-7677 cfp3112 wPt-10005 wPt-10012 wPt-10080 wPt-10230 wPt-10277 wPt-10618 wPt-10633 wPt-10634 wPt-10640 wPt-10958 wPt-10970 wPt-1191 wPt-1349 wPt-1406 wPt-1484a wPt-1484b wPt-2936 wPt-3046 wPt-4431 wPt-6216 wPt-6467 wPt-6742 wPt-8434 wPt-8686 wPt-8990 wPt-9106 wPt-9250 wPt-9579 wPt-10948 wPt-8579 wPt-0672 wPt-10389 wPt-10420 wPt-10552 wPt-10712 wPt-10969 wPt-1612 wPt-2720 wPt-4777 wPt-5244 wPt-5495 wPt-8126 wPt-9088 stm6tcac stm537acag wPt-10871 rPt-7228 wPt-0264 wPt-0325 wPt-10171 wPt-10221 wPt-10330 wPt-11025 wPt-11252 wPt-11367 wPt-11454 wPt-2045 wPt-2557 wPt-3488 wPt-3529 wPt-4035 wPt-4403 wPt-6132 wPt-6211 wPt-7668 wPt-6239 wPt-10629 wPt-11347 cfp1773 tPt-1366 tPt-1759 wPt-10289 wPt-10325 wPt-10433 wPt-10485 wPt-10687 wPt-10688 wPt-11117 wPt-11286 wPt-11320 wPt-11341 wPt-11445 wPt-11451 wPt-1439 wPt-2404 wPt-3234 wPt-3626 wPt-3635 wPt-4630 wPt-4954 wPt-5105 wPt-6945 wPt-6973 wPt-7015 wPt-7260 wPt-7564 wPt-7737 wPt-8718a wPt-9170 wmc231 Iha-B1.2 ksuB8 psr902 barc68 gwm284 gwm566 wPt-10524 cfp1329 cfb3260 dw724 wmc505 wmc777 wPt-1081 wPt-2757 wPt-3094 wPt-6952 wPt-9351 wPt-1159 wPt-10642 wPt-3079 wPt-4209 67.1 67.3 67.6 67.9 68.4 69.1 69.3 69.7 70.3 70.6 70.7 70.8 71.3 72.3 73.2 74.4 74.6 74.8 74.9

66.9

62.4 62.9 63.5 63.6 64.1 64.4 64.7 64.8 65.5 65.6 65.9 66.4 66.8

62.2

61.9 62.0 62.1

61.6

61.5

61.3

61.2

58.1 59.3 60.3 60.5 60.7

58.0

56.5 57.0 57.1

56.4

54.7 54.8 55.0 55.8

54.4

54.0

50.3 50.6 50.7 51.0 51.3 51.5 51.7 52.8 53.0 53.1 53.5 53.7

wPt-10130 wPt-10409 wPt-10886 cfp3275 wPt-1158 wPt-2890 wPt-11404 wPt-1514 wPt-10196 wPt-10669 wPt-4006 wPt-7486 wPt-9573 wPt-10530 cfp5129 wPt-10458 wPt-10955 wPt-11440 wPt-1271 wPt-9510 wPt-1386 barc234 wPt-5390 wPt-5593 wPt-0371 wPt-10496 wPt-10756 wPt-5670 wPt-10008 wPt-10285 wPt-10540 wPt-10677 wPt-10690 wPt-11427 wPt-7403 wPt-11295 wPt-1948 wPt-5939 wPt-8915 wPt-9323 wPt-9432 wPt-4846 wPt-0327 wPt-6905 wPt-7152 wPt-11160 cfp5109 cfb3266 wmc612 wmc625 wmc762 cfp1417 cfp161 barc1111 gpw1145 nw2715 wmc366 wmm1007 wmm805 rPt-5396 wPt-0682 wPt-10961 wPt-1846 wPt-2091 wPt-4127 wPt-4608 barc139 cfb3409 gwm285 wPt-11032 wPt-4807 barc73 gwm376 wPt-2377 wPt-1838 wPt-6395 wPt-9226 wPt-11140 wPt-11248 wPt-2674 wPt-8136 wPt-9879 wPt-10319 wPt-10724 wPt-11224 wPt-11230 wPt-11364 wPt-11464 wPt-2126 wPt-3124 wPt-6319 wPt-10615 wPt-10682 wms285 wPt-10732 wPt-5068 wPt-7568 wPt-8733 wPt-8912 wPt-8738 wPt-11407 wPt-11480 wPt-0985 wPt-10658 wPt-10661 wPt-11116 wPt-5300 wPt-6047 barc1044 wPt-10323 wPt-10422 wPt-10894 wPt-4048 wPt-6020 wPt-7388 cfb3135 wPt-10490 wPt-10507 wPt-10553 wPt-7511 wPt-8512 wPt-8663 wmm723 wPt-4597 wPt-1625 wPt-5716 wPt-7229 wPt-9310 dw1221 wPt-10033 wPt-10042 wPt-10514 wPt-10542 wPt-10609 wPt-10697 wPt-10848 wPt-10885 wPt-11143 wPt-11237 wPt-11476 wPt-1839 wPt-1904 wPt-2002 wPt-2235 wPt-3478 wPt-5282 wPt-6332 wPt-6699 wPt-7657 dw1754 wPt-11024 wPt-11325 wPt-2184 wPt-9433 wPt-2458 wPt-7142 wPt-11218 wPt-3078 wPt-9443 wPt-7692 wPt-8150 stm405tgag stm5tgag wmc307 wPt-0446 wPt-0544 bcd809 stm516tcac barc251 tPt-5281 cfb3374 dw955 wPt-2220 wPt-5058 ksuG59 cfp1632 nw1666 Pc-A12 wPt-10497 wPt-10875 wPt-11254 wPt-11392 wPt-1940 wPt-3327 wPt-4364 wPt-5319 wPt-5457 wPt-7786 wPt-7502 wPt-10003 wPt-2372 wPt-0212 gpw4034 wmc418 cfp100 wPt-9012 barc164 barc358 wPt-5024 wmc471 wPt-10142 wPt-10560 wPt-9367 nw2706 wmm1703 cdo718 wmc334 wPt-4082 wPt-9538 wPt-2280 wPt-5786 wPt-10462 wPt-7095 wPt-11324 gwm383 wPt-1171 wPt-0644 wPt-10294 wPt-7688 wPt-10358 wPt-0124 wPt-11177 wPt-10179 wPt-11219 wPt-1376 wPt-5452 wPt-7025 cfp3041 wPt-2698 wPt-9049 wPt-1804

90.0 90.7 90.9 91.3 91.6 91.8

99.4

97.2

94.0 94.2

cfa2170

wPt-10030 wPt-10532 wPt-10644 wPt-10786

wPt-10176 nw1608

wPt-10170

89.5

83.2 83.3 83.5 83.6 84.5 84.8 84.9 85.5 86.6 86.8 87.1 87.8 88.0

82.7

77.4 78.3 78.6 78.8 79.4 79.8 79.9 80.9 81.3 81.7 82.1 82.2 82.4 82.5

77.0

wPt-7254 cfb3059 wPt-6376 wPt-11378 wPt-9169 tPt-7068 wPt-10832 wPt-1754 wPt-5020 wmm1385 wPt-9912 tPt-3599 wPt-10270 wPt-10437 wPt-10442 wPt-10600 wPt-11304 wPt-7145 wPt-8964 cfp1343 cfb59 wPt-10546 wPt-11400 wPt-5329 wPt-5906 wPt-8096 wmc291 wPt-2710 barc229 gwm108 wPt-11278 wPt-2015 wPt-3107 wPt-10411 wPt-1208 wPt-8056 wPt-10384 wPt-10834 wPt-11028 wPt-6187 wPt-10276 wPt-11171 wPt-10455 wPt-11299 wPt-1622 wPt-7635 wPt-0384 wPt-0036 wPt-8480 wPt-8324 wPt-0065 wPt-10188 wPt-10293 wPt-10653 wPt-10785 wPt-11053 wPt-11301 wPt-1945 wPt-4697 wPt-5769 wPt-7732 wPt-9026 wPt-9232 wPt-4222 wPt-10913 cfb3440 wmm1133 wPt-7968 wmm702 cfp39 wPt-11303 wPt-5833 wPt-8318 wPt-3678 wPt-2904 wPt-7436 wPt-0896 P39/M50-3 wPt-6785 wPt-4428 wPt-10279 wPt-11004 wPt-5640 wmm280 wPt-5358 wmm278 wPt-3005

75.2 75.3 75.6 75.8 75.9 76.5

124.1

wmm448

145.4

144.2

143.1

141.3 141.4 141.7 142.0

141.2

140.5

140.3

140.2

140.1

139.9 140.0

139.7

139.5

139.4

133.8 133.9 134.3 134.6 134.9 136.4 136.7

133.1

132.6

131.7 132.2

131.6

128.0 128.2 128.4 128.9 129.3 130.0 130.1 130.7 131.0 131.5

127.6

127.1

125.8 126.2 126.5

tPt-1093 wPt-10000 wPt-10025 wPt-10240 wPt-10428 wPt-10555 wPt-10646 wPt-10829 wPt-10890 wPt-11082 wPt-11088 wPt-11330 wPt-2439 wPt-3153 wPt-4194 wPt-4517 wPt-5261 wPt-5268 wPt-5947 wPt-7802 wPt-8559 wPt-9185 wPt-9766 wPt-3791 wPt-10537 wPt-11452 wPt-4085 wPt-0367 wPt-0401 wPt-10675 wPt-11221 wPt-11235 wPt-1870 wPt-2177 wPt-2391 wPt-2731 wPt-4570 wPt-5295 wPt-7352 wPt-8021 wPt-9368 wPt-0912 wPt-11044 wPt-11350 wPt-1153 wPt-1458 wPt-7098 wPt-8718b wPt-8959 wPt-10349 wPt-1884 wPt-3342 wPt-4493 wPt-6856 psr931 wmc236a wPt-1108 wPt-4933 wPt-2685 wPt-0021 wPt-10039 wPt-4401 gwm114 wPt-3760 wPt-10157 wPt-10341 wPt-10352 wPt-10366 wPt-10582 wPt-10587 wPt-11055 wPt-11372 wPt-11455 wPt-4483 wPt-4782 wPt-5501 wPt-6815 P36/M48-5 wPt-8845b wPt-11168b wPt-11292 wPt-5648 wPt-7097 wPt-8206 wPt-8438 rPt-0996 wPt-0492 wPt-0665 wPt-0940 wPt-10043 wPt-10326 wPt-11017 wPt-11228 wPt-11241 wPt-11314 wPt-1834 wPt-2851 wPt-3525 wPt-7514 wPt-8386 wPt-9989 wPt-5072 wPt-7614 wPt-9189 wPt-11182 wPt-8513 wPt-4808 wPt-7037 wPt-9357 wPt-0280 wPt-0365 wPt-0405 wPt-8141 wPt-8983 wPt-7595 tPt-7594 wPt-0324 wPt-0531 wPt-0668 wPt-1151 wPt-2559 wPt-3397 wPt-6131 wPt-7526 wPt-7705 wPt-7886 wPt-10758 wPt-10805 wPt-6834 wPt-10407 wPt-10502 wPt-10534 wPt-10541 wPt-10701 wPt-10731 wPt-10777 wPt-11103 wPt-11194 wPt-11276 wPt-11420 wPt-11443 wPt-1822 wPt-2784 wPt-4370 wPt-4576 wPt-5825 wPt-7357 wPt-7956 wPt-0751 wPt-7158 wPt-10071 psr454 psr604 wPt-4412 wms340 wPt-3856 wPt-10874 wPt-11306 wPt-1219 wPt-5965 wPt-8355 gwm247 wPt-4410 wPt-7266 wPt-7627 wPt-8396 tPt-4541 wPt-0244 wPt-0855 wPt-1020 wPt-10201 wPt-10505 wPt-6961 wPt-7340 wPt-9694 wPt-9826 wPt-2416 wPt-0438 wPt-0995 wPt-10052 wPt-10729 wPt-2567 wPt-3410 wPt-6008 wPt-7733 wPt-8352 wPt-8643 wPt-9495 wPt-1311 gpw4513 wPt-2119 wPt-5580 wPt-5946 wPt-11155 wPt-2277 wPt-3165 wPt-5033 wPt-6113 wPt-7060 wPt-7741 wPt-1977 wPt-1033 wPt-10453 wPt-10941 wPt-11144 wPt-2104 wPt-9228 wPt-9795

Long arm

gpw3233 123.4

122.5

121.9

wmm274 wPt-0533 wPt-0875 wPt-10065 wPt-10077 wPt-10464 wPt-10626 wPt-11428 wPt-2020 wPt-3822 wPt-5430 wPt-6299 wPt-6407 wPt-7908 wPt-8184 wPt-8752 wPt-0990 wPt-10303 wPt-10589 wPt-11000 wPt-11253 wPt-4225

wPt-7392

wmm1966

cfb22 cfb3200 wPt-11029 barc77

P36/M47-2 wPt-5704 cfp24 wmm430 wPt-4974

wmc326 wPt-0345 wPt-11050 wPt-11300 wPt-11413 wPt-1275 wPt-2491

wPt-11049

wPt-7301 wPt-6436

P37/M47-7 stm625tcac

121.3

120.3

117.8

116.4 116.7

114.9 115.2 115.6

113.3

108.0

107.2 107.5

100.6

125.6

Fig. 3 Consensus map of chromosome 3B presented from top (short arm) to bottom (long arm), as derived from 20 individual maps built for 19 DH, RIL, and F2 populations. DArT markers are in black; SSR and other markers are labeled in red (see ESM1 for details)

14.2 14.4 14.6 15.1 15.7 16.8 16.9 18.9 19.2 21.5 22.1

14.0

13.5 13.7 13.8

13.3

13.2

13.1

12.9

12.6 12.7

12.2

gwm389 wPt-10593 wPt-1620 wPt-7264 wPt-8093 gwm533 stm512tcac stm559tgag stm598tcac wPt-1162 wPt-1542 cfp3132 cfp140 wPt-8446 wPt-5625 wPt-5209 wPt-5522 wPt-7225 cfb3296 cfb3530 glk683/Sun2 stm599tcac wPt-7341 wPt-11068 wPt-5045 cfb3417 swm13 wmm756 wPt-11391 wPt-7984 wPt-2748 wPt-11419 gwm533 wPt-10192 wPt-10239 wPt-10503 wPt-10910 wPt-10982 wPt-8646 wPt-10291 wPt-10388 wPt-6129 wPt-10972 wPt-11346 wPt-1867 wPt-3704 wPt-6813 wPt-10272 wPt-10282 wPt-10826 wPt-10850 wPt-10921 wPt-11331 wPt-11390 wPt-11414 wPt-1540 wPt-2621 wPt-2849 wPt-3260 wPt-3609 wPt-3921 wPt-4224 wPt-4437 wPt-6291 wPt-7134 wPt-8079 wPt-9674 barc238 wPt-10186 wPt-11225 wPt-11388 wPt-1935 wPt-2241 wPt-4860 wPt-5521 wPt-5579 wPt-5622 wPt-7342 wPt-7504 wPt-7907 wPt-9503 wPt-3761 wPt-10006 barc133 wPt-10710 wPt-10884 wPt-11369 wPt-1391 wPt-1971 wPt-3550 wPt-3943 wPt-9670 wPt-10333 wPt-7961 cfb5010 wPt-10311 wPt-10940 wPt-11111 wPt-11305 wPt-3185 wPt-9789 gpw7757 wPt-6718 tPt-9267 barc147 stm560tgag wPt-10372 wPt-10538 wPt-10614 wPt-11279 wPt-8411 cfb6012 wPt-9703 wPt-0302 wPt-10714 wPt-3536 wPt-10818 wPt-9066 wPt-3598 wPt-4648

stm513tctg stm538acag

4.5

8.4 8.7 9.0 9.2 9.4 9.5 10.2 10.5 10.6 10.8 10.9 11.3 11.9 12.0 12.1

wPt-2368 wPt-9496

0.0

Short arm

Theor Appl Genet (2010) 121:465–474 471

123

472

Theor Appl Genet (2010) 121:465–474

Table 2 Genetically distinct loci on the wheat chromosome 3B consensus map as identified by different subsets of markers Data set

Marker source

No. of individual maps in consensus map

No. of markers

No. of unique loci

DArT ? SSR

Whole genome ? 3B

20 (whole-genome DArTs), 4 (3B DArTs), 5 (SSR)

939a

304

Whole genome

4

186a

92

3B (flow-sorted)

4

510

132

Whole genome ? 3B

4

696a

192

Whole genome

20

269a

142

Whole genome ? 3B

20 (whole genome), 4 (3B)

779a

237

Whole genome

5

160

101

DArT

b

SSRb a

One marker (wPt-8718) was mapped to two different loci at 49.7 and 127.6 cM

b

DArT markers were developed in a parallel fashion by randomly drawing DNA fragments from genomic representations. They were not filtered for redundancy, for example by sequencing. In contrast, SSR markers were developed individually in a serial manner, a process during which redundancy is eliminated

This observation confirmed the reproducibility of flow karyotyping in hexaploid wheat. Flow-sorted chromosomes were identified by FISH with two different DNA probes, which turned out to be useful approach. While the use of GAA microsatellite and Afa as probes allowed the identification of chromosomes 3B and 1BS, in situ probing with a labeled probe for the telomeric repeat unambiguously identified intact 1BS telocentrics, thereby avoiding confusion of 1BS with chromosome arms of similar size generated by breakage of other chromosomes in the process of sample preparation and sorting (Kubala´kova´ et al. 2002). As less than 5 ng of DNA is sufficient to prepare genomic representations, there was no need for chromosomal DNA amplification. Direct use of isolated DNA to develop DArT markers eliminated any potential bias due to amplification, and preserved the DNA methylation status, which is important since the DArT protocol is based on the utilization of methylation-sensitive restriction enzymes. Thus, the requirement for non-amplified DNA excludes the use of chromosome microdissection in the DArT process, which in any case can only be applied to a small number of chromosomes (Hobza and Vyskot 2007). At the same time, a single batch of flow-sorted chromosomes (prepared within 1 h) is sufficient to selectively develop DArT markers from a chromosome and/or chromosome arm of interest. Efficiency of chromosome-specific DArT marker development Some markers derived from chromosome arm 1BS and chromosome 3B mapped to other genome regions, particularly to chromosomes belonging to the B genome (1BS: 5 of 9 markers or 55%; 3B: 68 of 158 markers or 43%). Several reasons may account for the fact that not all DArT markers developed from chromosomal DNA mapped to the source chromosomes. First, the purity of flow-sorted chromosome fractions only rarely reaches 100%, as these

123

fractions are to some extent contaminated by other chromosomes (Dolezˇel et al. 2007). For chromosome 3B, this B-genome preference could be explained by a slight contamination of the chromosome 3B fraction by other B-genome chromosomes, which constitute the majority of chromosomes in peak III next to the 3B peak (Kubala´kova´ et al. 2002) (Fig. 1). The 1BS fraction could preferentially be contaminated with chromatids of chromosomes 1D, 4D, and 6D with relative fluorescence intensity similar to 1BS and/or short arms separated from intact chromosomes with molecular sizes similar to 1BS, such as 4AS, 6AS, 5BS, 2DS, 3DS, and 6DS. In fact, there is a striking similarity between the average purity of sorted 1BS fractions (85.5%) and the proportion of 1BS-derived markers mapping to chromosome 1B (86.7%). A larger difference was observed for chromosome 3B, where the average purity of the sorted fractions reached 90.8%, whereas ‘only’ 77.8% of 3B-derived markers mapped to chromosome 3B. The factors reducing the yield below the expected yield are unknown, but may be related to the size of chromosome 3B. Although most DArT markers reported a single locus, a minority of them reported an alternative locus on homoeologous chromosomes or repeated regions of the genome. The chance of cloning such markers should be greater for 3B than for other chromosomes, simply because with a size of 993 Mbp, 3B is the largest chromosome of wheat. With 78–87% of markers developed from sorted chromosomes mapping to the original chromosomes, our strategy for targeted development of DArT markers appears quite efficient and holds a considerable promise to significantly increase the number of markers for a chromosome (or chromosome arm) of interest. The enrichment advantage is expected to be most pronounced in large genomes with many chromosomes such as wheat. With a molecular size of 314 Mbp, chromosome arm 1BS represents only 1.9% of the hexaploid wheat genome. With 86.7% of markers developed from the sorted arm being specific for 1B, the efficiency is 45-fold higher relative to

Theor Appl Genet (2010) 121:465–474

the whole genomic DNA approach. A 13-fold increase in efficiency was achieved for chromosome 3B, the largest wheat chromosome 3B (993 Mbp), which represents 5.8% of the genome. A consensus map for chromosome 3B As shown in Table 2, DArT markers cloned from wholegenome DNA preparations covered 142 loci, while those derived from flow-sorted 3B chromosomes covered 132 loci. The latter comparison is skewed against the 3B-derived markers for two reasons. First, the 3B-derived DArT markers were only genotyped in four populations, while the whole genome-derived DArT markers were assayed across 20 populations, thereby increasing their chance of being polymorphic in at least one of the parental combinations. Second, the 3B-derived markers were prepared from only five different wheat cultivars (see ‘‘Materials and Methods’’; Table 1), while whole genomederived markers were cloned from 234 different cultivars and accessions. The much narrower genetic diversity of accessions from which 3B-specific (and even more in case of 1BS-specific) markers were derived resulted in cloning fewer unique fragments corresponding to DArT alleles scored as ‘‘present’’ when compared to the ‘‘whole genome’’ approach, where we selected a broad range of genotypes to more broadly represent wheat genetic diversity. This lower number of unique cloned fragments limited the number of genetic loci that could be covered in this approach.

Conclusions In this study, we demonstrate that the DArT platform can be coupled with chromosome sorting to target specific regions of a large genome to significantly increase the saturation of linkage maps of a chromosome of interest. Our pilot study on hexaploid wheat shows that DArT markers can be developed from DNA of sorted chromosomes at a 13–45-fold increased efficiency compared to DArTs developed from genomic DNA. More than 600 new markers were developed for wheat chromosome 3B and chromosome arm 1BS. The number of genetically distinct loci on the 3B consensus map was more than doubled. Because each of the 42 chromosome arms can be isolated by flow-sorting (Kubala´kova´ et al. 2002), any chromosome can now be saturated with a large number of DArT markers. The physical map of wheat is being constructed using a chromosome-by-chromosome approach, where individual laboratories develop maps for individual chromosomes (Feuillet and Eversole 2008). These laboratories can now develop saturated DArT maps for their

473

specific chromosomes in an affordable and targeted manner. In principle, this approach is applicable to any crop species for which methods of chromosome sorting are available (Dolezˇel et al. 2004). Thus, DArT markers from flow-sorted chromosomes further increase the utility of the platform and provide a significant complement to other marker technologies in genetic, genomic, and breeding applications. Acknowledgments We thank Dr. Jarmila Cˇ´ıhalı´kova´ and the data production team at Diversity Arrays Technology Pty. Ltd. for excellent technical assistance. This work was supported by research grant no. LC06004 from the Czech Ministry of Education, Youth and Sports and the Grains Research and Development Corporation (GRDC) of Australia.

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