Detection of sequences conferring resistance to cephalosporium stripe disease in wheat via in situ hybridization with a thinopyrum-specific transposon probe
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Authors
Newman, Peter D. and Gomez, Christian L.
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Type
Language
eng
Keywords
Cephalosporium stripe disease, Wheat, Fungus, Pathogens
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Abstract
The fungal pathogen Cephalosporium stripe disease attacks ceral crops, including wheat and can cause a loss of over 50% in yield due to decreased seed size and reduced seed production. We investigated Cephalosporium-resistant wheat lines developed at Washington State University. The pedigrees of these wheat lines incorporated parents from the wheatgrass Thinopyrum, which has an ability to withstand a variety of crop diseases including Cephalosporium stripe disease. We hypothesize that sequences retained from the Thinopyrum lineage are responsible for the disease resistance in these lines, and were incorporated into the wheat genome through chromosomal translocation or DNA crossover. We isolated a Thinopyrum-specific transposon sequence from wheat-Thinopyrum amphiploids. This transposon was fluorescently-labeled and employed as a probe for in situ hybridization to detect regions of Thimopyrum-derived chromatin in the resistant wheat lines WA7970, WA7971, and WA8000 as well as in control lines. Our use of a genome-specific transposon probe is intended to provide greater binding specificity than our previous methods employing fluorescently-tagged whole genomic DNA, which can bind to common sites on both Thinopyrum and wheat chromosomes.
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ABSTRACT BACKGROUND METHODS Chromosome Prep & in situ Hybridization 3-day old root meristem tissue was pre-fixed for 24 hours at 0.1°C, then fix for 48 hours in 3:1 ethanol:acetic acid and squashed under a cover glass. Slides were frozen at -80 °C then dehydrated in 45% acetic acid and 95% ethanol and chromosomal DNA was denatured by incubation in 70% formamide at 70°C for 2 minutes. Genomic DNA probe was prepared using the BioNick nick translation system (Invitrogen). Blocking DNA was prepared by autoclaving Chinese Spring genomic DNA till it sheared to length of 200-500 nt. A hybridization solution of 25% dextran sulfate, 60% formamide, 0.2X SSC, 50 ng of biotinylated probe and 2.5 μg of blocking DNA was applied to each slide and incubated for 16 hours at 37°C. Excess probe was removed through a rinse in 2X SSC. Slides were incubated in a solution of avidin-fluorescin, followed by biotinylated anti-avidin and a second incubation in avidin-fluorescin. Slides were washed thrice with 4XSSC-Tween20 between treatments. Preparations were counterstained with 10 μg/mL propidium iodide. gDNA Extraction & Transposon Isolation Fresh leaf tissue was ground in liquid nitrogen and the resulting powder was incubated in a solution of 100 mm Tris, 500 mm NaCl, 20 mm EDTA, 1.25% SDS and 1% sodium bisulfite at 65 C for 30 minutes. An equal volume of 24:1 chloroform:isoamy-alcohol was added and the samples were centrifuged at 4500 rpm for 15 minutes. The aqueous layer was removed and DNA was precipitated with cold isopranol, rinsed with 70% and 95% EtOH, dried, and resuspended in 1 mL 1X TE Buffer with 10 mg/mL RNaseA. The PCR solution consisted of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 200 μM dNTP, 1.0 U Taq polymerase (NEB) and 25 pmol each of primers ple2F and ple2R in a 20 μL reaction. The reactions were carried out over 35 cycles of 94°C for 60 seconds, 47°C for 45 seconds and 72°C for 45 seconds. 5 μL of the PCR product was loaded into a 1.0% agarose gel and run at 100V; the resulting 277 A:T-rich transposon sequences were excised and purified using the QiaQuick Gel Extraction Kit (Qiagen), following manufacturer’s protocols. PCR of the isolated tranposon sequence was repeated after gel extraction, as described above, but substituting the dNTP solution with one containing a mixture of 0.1M dATP and 0.1M biotin-14-dATP. CYTOLOGY RESULTS pLe2 TRANPOSON ISOLATION CONCLUSIONS & FUTURE DIRECTIONS PEDIGREE INFORMATION DETECTION OF SEQUENCES CONFERRING RESISTANCE TO CEPHALOSPORIUM STRIPE DISEASE IN WHEAT VIA IN SITU HYBRIDIZATION WITH A THINOPYRUM-SPECIFIC TRANSPOSON PROBE Peter Newman, Christian Gomez & Matt Arterburn, Biology Dept, Washburn University WA7970 Chinese Spring Wheat Rush Wheatgrass (Th. ponticum) Perennial Hybrid Amphiploid WA7971 Pawnee (ca. 1943) HARD WHITE WHEAT Omar Wheat (ca. 1955) (BACKCROSSED TWICE) SOFT WHITE CLUB WHEAT Madsen (ca. 1989) BACKCROSSED TWICE Madsen (ca. 1989) Paha (ca. 1972) HARD RED WHEAT Table 1. Cephalosporium stripe disease index and % seed infected by Cephalosporium gramineum for four winter wheat varieties grown at the Palouse Conservation Field Station, Pullman, 2006.* Variety/Line % infected seed Disease index Eltan 0.31 43.7 Madsen 0.46 67.8 Stephens 0.26 72.3 WA7970 0.20 37.7 * Murray, TD and SS Jones. 2007. REFERENCES Li HJ, Arterburn M, Jones SS, Murray TD. 2005. Resistance to eyespot of wheat, caused by Tapesia yallundae, derived from a Thinopyrum intermedium homoeologous group 4 chromosome. Theoretical and Applied Genetics. August. p. 1-9. Murray, TD and SS Jones. 2007. Resistance to and Seed Transmission of Cephalosporium Strip in Wheat. O.A. Vogel Wheat Research Fund Progress Report. Cephaloporium Stripe Disease Symptoms in Winter Wheat The fungal pathogen Cephalosporium gramineum attacks cereal crops, including wheat and can cause a loss of over 50 percent in yield due to decreased seed size and reduced seed production. We investigated Cephalosporium-resistant wheat lines developed at Washington State University. The pedigrees of these wheat lines incorporated parents from the wheatgrass Thinopyrum, which has an ability to withstand a variety of crop diseases including Cephalosporium stripe disease. We hypothesize that sequences retained from the Thinopyrum lineage are responsible for the disease resistance in these lines, and were incorporated into the wheat genome through chromosomal translocation or DNA crossover. We isolated a Thinopyrum-specific transposon sequence from wheat-Thinopyrum amphiploids. This transposon was fluorescently-labeled and employed as a probe for in situ hybridization to detect regions of Thinopyrum-derived chromatin in the resistant wheat lines WA7970, WA7971 and WA8000 as well as in control lines. Our use of a genome-specific transposon probe is intended to provide greater binding specifity than our previous methods employing fluorescently-tagged whole genomic DNA, which can bind to common sites on both Thinopyrum and wheat chromosomes. FUNDING This work was made possible through the Washburn University Transformational Experience Schlolarly/Creative grant fund. The fungal pathogen Cephalosporium gramineum attacks wheat and other cereal crops like rye and winter barley. Wheat is the major economic host for this fungal disease, which can cause a loss of over 50 percent in yield due to decreased seed size and reduced seed production. First reported in North America 1955, Cephalosporium stripe disease has been found in all Midwestern and Northwestern states. Currently, many crop varieties lack a resistance to this fungus. Researchers from the Winter Wheat Breeding lab at Washington State University developed wheat lines that incorporate wheatgrass parents from the genus Thinopyrum. Thinopyrum wheatgrass has an ability to withstand a variety of crop diseases including Cephalosporium stripe disease. These developed winter wheat lines also exhibit resistance against C. gramineum (Table 1). It is hypothesized that the Thinopyrum genes for disease resistance were incorporated into the wheat genome, through chromosomal translocation or DNA crossover. To investigate our hypothesis, we conducted fluorescent genomic in situ hybridization (FGISH) on metaphase chromosomes from three advanced breeding lines that have Thinopyrum parents in their lineage Based on our initial results we are developing an assay using FISH with a Thinopyrum-genome specific transposon as a probe. Our expectation is that this specific probe will identify the expectedly narrow portion of foreign chromatin in these genomes with greater specificity. Biotin Transposon probe Chromosome Biotinylated anti-Avidin Ab Fluorescinated (Green) avidin Excise & purify DNA Fragment with Gel Extraction Kit Repeat PCR with labeled biotin-14-dATP & use probe for in situ hybridization PCR of wheat/wheatgrass hybrid with ple2 5’ & 3’ primers 277 bp • Our initial FGISH results failed to detect any “hotspots” of probe-binding, despite strong binding in controls. Non-specific hybridization was markedly increased over controls, possibly due to a lack of high-association binding sites. • Thinopyrum-specific transposon pleUCD2 was chosen as an alternate probe. Binding efficiency of a ple2 probe should be increased over gDNA probes since there are many potential common-binding sites between wheat and wheatgrass when employing the latter. • ple2 sequences were successfully amplified using PCR from wheat-Thinopyrum amphiploids and chromosome addition lines. The resulting PCR product was isolated and purified for biotin-14-dATP labeling. • No detectable ple2 PCR product was amplified from WA7970, WA7971 or WA8000. This is, however, not diagnostic because the weak amplification of ple2 (the entire sequence is highly A:T rich) generally prevents amplification of chromatin in quantities lower than what is found in a monosomic Thinopyrum addition line (Li et al, 2004). WA7970 WA7971 WA8000 PERENNIAL HYBRID Phase Contrast Phase Contrast Phase Contrast CONTROL FGISH FGISH FGISH FGISH