Septoria and Stagonospora Diseases of Cereals - CIMMYT ...

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Identification of a Molecular Marker Linked to Septoria Nodorum Blotch Resistance in Triticum tauschii Using F2 Bulked Segregant N.E.A. Murphy, 1 R. Loughman, 2 R. Wilson, 2 E.S. Lagudah, 3 R. Appels, 3 and M.G.K. Jones1 1 WA State Agriculture Biotechnology Centre, Division of Science and Engineering, Murdoch University, Murdoch, Australia 2 Agriculture Western Australia, Bentley Delivery Centre, Western Australia 3 Plant Industries, CSIRO, Canberra, Australia Abstract A search was conducted for a molecular marker linked to a gene for resistance to septoria nodorum blotch in the Triticum tauschii accession RL5271. DNA was extracted from leaves of F2 plants which were progeny tested to identify homozygous resistant and homozygous susceptible F2 plants. The DNA from the homozygous resistant plants was pooled together and the low copy sequences were enriched using renaturation kinetics and hydroxyapatite to remove the repetitive DNA sequences. The pooled DNA from the homozygous susceptible plants was treated in the same manner. The pooled DNA and the parental DNA were screened using RAPD primers. Two markers present in the pooled resistant DNA and in the parental DNA were identified and cloned. These markers were verified using RFLPs with the cloned marker as a probe. One of the probes did not produce any polymorphism as a RFLP, even when a number of different restriction enzymes were used. The second marker was polymorphic between the two parents when the DNA was restricted with HindIII and then MseI. In the 14 homozygous F2 plants tested, the marker was completely linked to the resistance gene. This marker may be valuable in introgressing the resistance gene from T. tauschii into a commercial bread wheat cultivar. Septoria nodorum blotch is the major disease of wheat (Triticum aestivum L.) in the Western Australian wheat belt. It is caused by Stagonospora nodorum (Berk.) Castellani & E.G. Germano (teleomorph Phaeosphaeria nodorum (E. Müller) Hedjaroude). The preferred method of control is the use of resistant cultivars, but this has been limited in the past by the lack of major genes for resistance. In bread wheat inheritance of resistance to septoria nodorum blotch is frequently complex (Mullaney et al., 1982; Scharen and Krupinsky, 1978). The complex and additive nature of septoria nodorum resistance in bread wheats has made the selection for resistance difficult. Relatively small improvements in the overall resistance can be difficult to identify, as they can be masked by strong environmental effects. Selection of resistant plants is complicated by the association of resistance and both plant height and maturity (Rosielle and Brown, 1980; Scott et al., 1982). The use of a molecular marker would help overcome these problems by avoiding interpretation of phenotype under environmental influences (Allen, 1994). Furthermore, if the molecular marker is co-dominant, it will make it possible to identify an individual plant as homozygous for resistance without further progeny testing. This ability to identify homozygous 71 plants would be particularly useful in any program where extensive backcrossing would be required, such as the introgression of a resistance gene from wild wheats. The aim of this work was to identify a molecular marker that is linked to a single gene conferring resistance to septoria nodorum blotch identified in the accession RL5271 of the wild wheat Triticum tauschii. Materials and Methods A cross between the resistant accession RL5271 and the susceptible accession CPI110889 was progeny tested. The F2 plants were progeny tested using F3
72 Session 3B — N.E.A. Murphy, R. Loughman, R. Wilson, E.S. Lagudah, R. Appels, and M.G.K. Jones families to identify homozygous F2 plants. The DNA of eight homozygous resistant F2 plants and 14 homozygous susceptible F2 plants was selected for the analysis. Equal amounts of DNA from the homozygous resistant F2 plants were bulked together as was the DNA from the homozygous susceptible F2 plants. Each bulked sample and the DNA from each parent were divided into two aliquots. One aliquot was left untreated. The second aliquot was treated to enrich the proportion of low copy sequence DNA by removing the repetitive DNA sequences using hydroxyapatite after selective renaturation. The technique followed Eastwood et al. (1994). The double-stranded DNA was removed from the samples after a Cot value of 120 had been achieved (Smith and Flavell, 1975). The DNA was re-suspended in 0.1xTE buffer and diluted to a final concentration of 5ng/ml of DNA. RAPD reactions were carried out on a Perkin-Elmer PE9600. The random primers were produced by Operon Technologies (Alameda, Ca.). The RAPD products were electrophoresed on polyacrylamide gels, and the gels were stained using ethidium bromide with gel images recorded digitally using a Bio-Rad Gel Doc 1000 system. For each primer nine reactions were carried out: a negative control, sterile distilled water, was used instead of template DNA, the next four reactions used the total genomic DNA as template, and the final four reactions used the nonrepetitive DNA as template. If a polymorphism was identified, the polymorphic band was stabbed with a sterile syringe needle. The needle was washed in sterile distilled water which was used as template for a further RAPD reaction using the same primer and reaction conditions that had generated the polymorphism. The amplified fragment was ligated into the plasmid pGEM-T. The plasmids were transformed into E. coli cells by heat shock. The following day colonies were screened using PCR primers, and permanent cultures were established from insert-containing clones. The cloned polymorphic band was used as an RFLP probe to verify and determine the linkage of the potential marker to the resistance gene. Total genomic DNA of the parental accessions was digested with a number of restriction enzymes, singly and in combination, to find a restriction enzyme that would produce a polymorphism with the marker between the resistant and the susceptible parent. The marker band was prepared using an alkali- SDS minprep, double digested with NcoI and SacI, separated on an agarose gel and purified using agarase. The purified band was labeled using random nonamer primers with 32P as dCTP. The membranes were hybridized overnight, washed the following morning, and exposed to autoradiograph film for up to one week. When a suitable restriction enzyme had been selected, the DNA from the selected seven homozygous resistant F2 plants and seven homozygous susceptible F2 plants was digested, electrophoresed and blotted using an alkali capillary blot. The membranes were autoradiographed for up to two weeks. Results Sixty random primers were used to screen the DNA samples. Two polymorphic bands were identified by comparing the banding pattern of the resistant parent and the resistant bulk with the susceptible parent and the susceptible bulk. The first was amplified using primer OPJ6 and was approximately 770bp long (OPJ6770 ). The second polymorphic band was amplified using primer OPJ18 and the fragment was approximately 350bp (OPJ18350 ). Both bands were ligated into pGEM-T. For the first marker, OPJ6770 , no restriction enzyme nor combination of enzymes tested was able to produce a polymorphism between the resistant and the susceptible parent. Consequently the marker could not be tested for its linkage to resistance. The second marker, OPJ18350 , produced a polymorphism between the resistant and susceptible parent when the DNA was double restricted with HindIII and followed by MseI. The polymorphism was present as an extra band in the resistant parent. All of the F2 plants tested were correctly classified as resistant or susceptible when the marker was used.
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Septoria and Stagonospora Diseases
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ii The Organizing Committee express
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iv 87 Genetic Variability in a Coll
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vi Foreword In the mid-1970s the id
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2 Opening Remarks — S. Rajaram ar
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4 Opening Remarks — S. Rajaram Ta
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6 Opening Remarks — S. Rajaram cu
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8 Opening Remarks — S. Rajaram br
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10 Opening Remarks — S. Rajaram T
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12 Opening Remarks — S. Rajaram t
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14 Opening Remarks — S. Rajaram C
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16 Opening Remarks — S. Rajaram r
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Session 1: Pathogen Biology Biology
Page 28 and 29: Table 2. The Septoria tritici/Stago
Page 30 and 31: Molecular Analysis of a DNA Fingerp
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 73 and 74: 66 Session 3B — E. Arseniuk and P
Page 75 and 76: 68 Cultivar variances Cultivar vari
Page 77: 70 Session 3B — E. Arseniuk and P
Page 81 and 82: 74 Inheritance of Septoria Nodorum
Page 83 and 84: 76 Session 3B — N.E.A. Murphy, R.
Page 85 and 86: 78 Session 4 — B.A. McDonald, C.C
Page 91 and 92: 84 Session 4 — E. Arseniuk, H.S.
Page 93 and 94: 86 Session 4 — C. Cowger, C.C. Mu
Page 95 and 96: 88 each isolate. Two of the probes
Page 97 and 98: 90 Mating Type-Specific PCR Primers
Page 99 and 100: 92 Session 4 — Bennett, R.S., S.-
Page 101 and 102: 94 Session 5 — M.W. Shaw and the
Page 103 and 104: 96 Session 5 — M.W. Shaw The path
Page 105 and 106: 98 Spore Dispersal of Leaf Blotch P
Page 107 and 108: Session 5 — C.A. Cordo, M.R. Sim
Page 109 and 110: 102 Epidemiology of Seedborne Stago
Page 111 and 112: Session 5 — D.A. Shah and G.C. Be
Page 113 and 114: 106 Session 6A / Session 6B — J.M
Page 119 and 120: Session 6A / Session 6B — C.C. Mu
Page 125 and 126: 118 Session 6C — M. van Ginkel an
Page 127 and 128: 120 Session 6C — M. van Ginkel an
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122 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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