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Molecular Analysis of a DNA Fingerprint Probe from Mycosphaerella graminicola S.B. Goodwin and J.R. Cavaletto USDA-ARS, Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN, USA Abstract Clones hybridizing to the Mycosphaerella graminicola DNA fingerprint probe pSTL70 were identified from subgenomic libraries and sequenced. Analyses of the DNA sequences of these clones plus the original pSTL70 clone revealed that pSTL70 contains part of the open reading frame for a probable homologue of an osmosensing histidine kinase gene from yeast. The remaining portion of the clone contained a partial reverse transcriptase gene sequence and a 29 base pair direct repeat, which could mean that the clone is a transposable element. Methods for converting transposable elements into improved DNA fingerprinting techniques are discussed. DNA fingerprinting is a powerful tool for analyzing the genetic structure of fungal populations. Several fingerprinting strategies have been employed, including those based on the polymerase chain reaction (PCR) such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and DNA amplification fingerprinting (DAF). These techniques can provide information on hundreds of potential genetic loci in a very short time. However, each method has problems that can limit its utility. The RAPD technique relies on annealing of short (only 10 base) primers at low temperatures. This leads to high variability and low transportability to other labs. AFLPs require a reasonably high degree of sophistication in expertise and facilities, and also can suffer from problems with repeatability. DAF is operationally simple but the large number of bands produced can be difficult to separate and interpret. The most widely used DNA fingerprint technique is restriction fragment length polymorphism analysis using small pieces of repetitive genomic DNA as probes. This technique has been used extensively to analyze the population biology of the ascomycetes Magnaporthe grisea (Hamer et al., 1989) and Mycosphaerella graminicola (McDonald and Martinez, 1991), and the oomycete Phytophthora infestans (Goodwin et al., 1992). Thousands of isolates of each species have been analyzed. The M. grisea repeat (MGR) 586 probe contains part of an inverted repeat transposon (Farman et al., 1996). However, the nature of the repeating elements in the M. graminicola pSTL70 and P. infestans RG57 probes has not been determined. This study was initiated to test whether the M. graminicola pSTL70 probe was part of a transposable element. The long-term goal is to clone individual DNA fingerprint loci and convert them to a PCR- 23 based system by designing specific primers to unique regions at each genetic locus. Materials and Methods Subgenomic libraries were constructed from isolates IPO 323 and 94269. These are the parent isolates of the M. graminicola mapping population (Kema et al., 1996). Approximately 2 µg of genomic DNA from each isolate was digested to completion with the restriction enzyme Pst I. DNA fragments from 0.5-3 kb and from 3-9 kb for each isolate were excised from gels and purified using Wizard PCR Prep (Promega, Madison, WI). The DNA fragments were then ligated into pBluescript vector and transformed into competent cells of E. coli strain INValphaF’. White colonies were transferred into 200 µL LB+amp medium in 96-well Microtest tissue culture plates and grown at 37°C overnight. The 96 cultures from each plate were transferred onto large (150 x 15 mm) LB+amp agar
24 Session 1 — S.B. Goodwin and J.R. Cavaletto plates using a replica plater, and incubated upside down at 37°C overnight. Colony lifts were made onto 8 x 12 cm pieces of Zeta Probe (BioRad) membranes by briefly laying the membrane pieces over the 96 colonies and lifting to pick up the bacteria. Membranes were placed colony side up onto blotting paper soaked with 10% SDS for 3 min, then 0.5 M NaOH/1.5 M NaCl for 5 min, 0.5 M Tris, pH 8.0/1.5 M NaCl for 5 min, and 6x SSC for 5 min. Finally, a UV Stratalinker (Stratagene) was used to crosslink the plasmid DNA to the membrane. For Southern analysis, pSTL70 DNA was labeled using the Random Primer Fluorescein labeling kit with antifluorescein- HRP (DuPont NEN) and hybridized according to the manufacturer’s instructions. Approximately 4,000 clones (2,000 from each isolate) were screened. Positive hybridizations were verified by digesting each positive clone with Pst I to release the insert, separating the fragments on agarose gels, blotting and probing as described above. Clones that hybridized after two rounds of screening were sequenced using a Pharmacia ALF automated DNA sequencer. Results Among 4,000 clones screened, five hybridized strongly to pSTL70. Complete sequences have been obtained for the original pSTL70 clone and three others. Clones 2E11, 9A5 and 11E8 each had large regions of near identity with pSTL70 (Table 1). However, the three clones shared no similarity with each other. BLASTX searches of the putative translation products identified a number of GenBank accessions with similarity to clones pSTL70, 2E11 and 11E8 (Table 1). The original pSTL70 clone had high similarity to a two-component regulator gene from yeast, Sln1, and a similar gene from Candida albicans. Clone 2E11 had even higher similarity to the same genes. This clone corresponded to and extended the 5’ end of pSTL70 (Figure 1). Clone 11E8 corresponded to and extended the 3’ end of pSTL70 (Figure 1). This sequence may code for a reverse transcriptase. The final 239 bases of pSTL70 contained part of the putative reverse transcriptase coding sequence, but not enough of the gene to obtain positive BLAST hits in GenBank. Table 1. Analysis of the Mycosphaerella graminicola DNA fingerprint clone pSTL70 and three additional clones that hybridized to pSTL70 in Southern analysis. Size Overlap Blastx E Clone (bp) with pSTL70 results value Comments pSTL70 2860 N/A Sln1 8e-14 29 bp repeat 2E11 3073 1278 Sln1 2e-15 Gene sequence 9A5 2640 1103 No hits N/A 29 bp repeat 11E8 1636 417 bp Reverse 0.006 Transposable transcriptase element (?) pSTL70 2E11 9A5 11E8 The first 917 bases of 11E8 are not related to the sequence of pSTL70. Clone 9A5 had no similarity to any sequences in GenBank. The first 1103 bases of this clone were identical to pSTL70, but the final 1537 bases were unrelated (Figure 1). Clones pSTL70 and 9A5 both contained a 29 bp sequence that was tandemly repeated approximately 3.5 times (Figure 1). Southern analysis of genomic DNA from the parents and several progeny isolates of the M. graminicola mapping population revealed that 9A5 also gave a DNA fingerprint pattern, while 2E11 and 16D7 did not (data not shown). Discussion The M. graminicola fingerprint probe pSTL70 (McDonald and Martinez, 1991) contains part of the open reading frame for the yeast two-component regulator gene Sln1. This gene has been shown to function as an osmosensing Sln 1 R RT Figure 1. Relationships among the Mycosphaerella graminicola DNA fingerprint probe pSTL70 and related clones. Regions of overlap are indicated by open boxes, unique regions by single lines. Clones are labeled on the left. Approximate locations of the Sln1 and reverse transcriptase (RT) open reading frames are indicated by arrows. The tandem repeats are indicated by R.
Page 1 and 2: Septoria and Stagonospora Diseases
Page 3 and 4: ii The Organizing Committee express
Page 5 and 6: iv 87 Genetic Variability in a Coll
Page 7 and 8: vi Foreword In the mid-1970s the id
Page 9 and 10: 2 Opening Remarks — S. Rajaram ar
Page 11 and 12: 4 Opening Remarks — S. Rajaram Ta
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Page 17 and 18: 10 Opening Remarks — S. Rajaram T
Page 19 and 20: 12 Opening Remarks — S. Rajaram t
Page 21 and 22: 14 Opening Remarks — S. Rajaram C
Page 23 and 24: 16 Opening Remarks — S. Rajaram r
Page 26 and 27: Session 1: Pathogen Biology Biology
Page 28 and 29: Table 2. The Septoria tritici/Stago
Page 32 and 33: histidine kinase in yeast, although
Page 34 and 35: According to the previous observati
Page 36 and 37: cultivars. The presence of pycnidia
Page 38 and 39: Provided that S. nodorum fungal gen
Page 40 and 41: ;; yy ;; yy 6 28% 1 32% ;y; ; y y ;
Page 42 and 43: Table 1. Sources of isolates or DNA
Page 44 and 45: Septoria/Stagonospora Leaf Spot Dis
Page 46: Interrelations among Septoria triti
Page 49 and 50: 42 Session 2 — B.M. Cunfer disper
Page 51 and 52: 44 Session 2 — B.M. Cunfer beyond
Page 53 and 54: 46 Aggressiveness of Phaeosphaeria
Page 55 and 56: 48 Session 2 — P. Halama, F. Lala
Page 57 and 58: 50 Growth of Stagonospora nodorum L
Page 59 and 60: 52 Session 3A — G.H.J. Kema and E
Page 61 and 62: 54 A Possible Gene-for-Gene Relatio
Page 63 and 64: 56 Diallel Analysis of Septoria Tri
Page 65 and 66: 58 Session 3A — X. Zhang, S.D. Ha
Page 67 and 68: 60 Session 3A — L. Gilchrist, C.
Page 69 and 70: 62 Session 3A — L. Gilchrist, C.
Page 71 and 72: 64 Session 3B — E. Arseniuk and P
Page 75 and 76: 68 Cultivar variances Cultivar vari
Page 79 and 80: 72 Session 3B — N.E.A. Murphy, R.
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74 Inheritance of Septoria Nodorum
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76 Session 3B — N.E.A. Murphy, R.
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78 Session 4 — B.A. McDonald, C.C
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84 Session 4 — E. Arseniuk, H.S.
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86 Session 4 — C. Cowger, C.C. Mu
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88 each isolate. Two of the probes
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90 Mating Type-Specific PCR Primers
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92 Session 4 — Bennett, R.S., S.-
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94 Session 5 — M.W. Shaw and the
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96 Session 5 — M.W. Shaw The path
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98 Spore Dispersal of Leaf Blotch P
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Session 5 — C.A. Cordo, M.R. Sim
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102 Epidemiology of Seedborne Stago
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Session 5 — D.A. Shah and G.C. Be
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106 Session 6A / Session 6B — J.M
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Session 6A / Session 6B — C.C. Mu
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118 Session 6C — M. van Ginkel an
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Session 6C — G. Shaner 128 An exa
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Session 6C — G. Shaner 130 Anothe
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Session 6C — M. Díaz de Ackerman
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134 Septoria tritici Resistance Sou
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Session 6C — L. Gilchrist et al.
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Session 6C — L. Gilchrist et al.
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140 Selecting Wheat for Resistance
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Session 6C — R.M. Gonzalez I., S.
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Session 6C — R.M. Gonzalez I., S.
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Session 6C — R. Loughman, R.E. Wi
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148 Field Resistance of Wheat to Se
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150 Using Precise Genetic Stocks to
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Session 6C — C.M. Ellerbrook, V.
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154 Evaluating Triticum durum x Tri
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156 Sources of Resistance to Septor
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Session 6C — H. Toubia-Rahme and
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160 Partial Resistance to Stagonosp
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Session 6C — C.G. Du, L.R. Nelson
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Session 6C — D.E. Fraser, J.P. Mu
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Session 6C — C.G. Du, L.R. Nelson
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Session 6C — M. Mincu 168 Arcsin
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170 Comparison of Greenhouse and Fi
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Session 6C — S.L. Walker, S. Leat
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Session 6D — L.N. Jørgensen, K.E
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176
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Concluding Remarks — Z. Eyal 178
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184 Dr. Patrice Halama Professor In
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186 Dr. Hala Toubia-Rahme Research
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