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TITLE:
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Identification of Three Genes for Powdery Mildew Resistance in a
Pm12 Wheat Line by Cosegregation Analysis and RAPD Markers
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AUTHORS:
A. N. Shi, S. Leath*, and J. P. Murphy
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ADDRESS:
Steven Leath
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Department of Plant Pathology
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North Carolina State University
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Raleigh, NC 27695-7616
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A. N. Shi, Dep. of Plant Pathology, North Carolina State Univ., Raleigh, NC 27695-7616;
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S. Leath, (USDA-ARS), Dep. of Plant Pathology, North Carolina State Univ., Raleigh,
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NC 27695-7616; J. P. Murphy, Dep. of Crop Science, North Carolina State Univ.,
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Raleigh, NC 27695-7629. Received_________________
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*Corresponding author (steven_leath@ncsu.edu).
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Abbreviations: Bgt, Blumeria graminis f. sp. tritici; Random amplified polymorphic
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DNA.
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ABSTRACT
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The reactions to six isolates, E314, 209a2, #8, 137a, 153a2, and Wkin91, of
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Blumeria graminis f. sp. tritici (Bgt) in the P1, P2, F1, F2, and BC1F1 generations derived
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from the cross between Line #31 containing the Pm12 resistance gene and a susceptible
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cultivar, NK-Coker 68-15, were analyzed. The resistance to E314 and 209a2 was controlled
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by three independent dominant genes, resistance to 137a1 and #8 was controlled by two
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independent dominant genes, and resistance to 153a2 and Wkin91 was controlled by one
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dominant gene in Line #31. An allelism test revealed the Pm12 wheat line also contained
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Pm1 based on the reaction to isolate 209a2 of Bgt in the P1, P2, F1, and F2 generations
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derived from the cross between Line #31 and Axminster/8*Cc with the single gene Pm1.
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Cosegregation of resistance to Bgt indicated that Line #31 contained three dominant genes.
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One was Pm1, another was Pm12, and the other was postulated to be Pm2. Four RAPD
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markers, OPAH05580, OPAI13490, OPAE12495, and OPAE121350, were found to be linked to
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Pm12, and the recombination frequencies between the four markers and Pm12 was 1.6 
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0.03 %, 6.6  0.10 %, 4.9  0.08 %, and 3.3  0.05 %, respectively. The RAPD marker
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OPAU17750 was found to be tightly linked to Pm1 in Line #31. The recurrent parent
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cultivar Wembley was postulated to contain Pm1 by host-pathogen interaction and linked
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RAPD marker analysis.
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1
Resistance to wheat powdery mildew (Blumeria graminis (DC.) E.O. Speer f. sp.
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tritici Em. Marchal, (Bgt) = Erysiphe graminis DC. ex Merat f. sp. tritici Em. Marchal)
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was introgressed into the UK spring wheat cultivar Wembley from Aegilops speltoides
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(2n=2x=14, SS) to produce Line #31 (Miller et al., 1988). Miller et al. (1988) reported
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that Line #31 contained a single dominant resistance gene, designated as Pm12, and
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located on chromosome 6A by monosomic analysis. Jia et al. (1996) concluded Pm12
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was located on the short arm of the translocation chromosome 6BS-6SS.6SL by use of
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RFLP-based maps of the homoeologous group-6 chromosome of wheat. It is possible
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there is more than one gene for powdery mildew resistance in Line #31 because the Bgt
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isolates used in their research were not clearly characterized and the mode of inheritance
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for resistance to differential Bgt isolates has not been analyzed in Line #31. Therefore, it
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is important to identify the genetic basis for wheat powdery mildew resistance in Line
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#31, since Line #31 was resistant to all tested isolates in Europe and the USA (Miller et
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al., 1988; Niewoehner and Leath, 1997).
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Molecular markers have been a very useful tool for detecting linked genes and for
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marker assisted selection (Stuber, 1992; Michelmore, 1995). RFLP markers linked to the
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gene Pm12 in Line #31 have been reported (Jia et al., 1996). We found RAPD marker
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OPU17750 tightly linked to the Pm1 gene was also present in the wheat Line #31, and
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postulated that Line #31 contained the Pm1 gene (Chapter 3). The objective of this
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research is to identify the mode of inheritance and genes for wheat powdery mildew
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resistance in Line #31 and to identify RAPD markers linked to Pm12 and Pm1 in Line
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#31. Concurrently, we wished to improve a method to identify more than one gene for
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powdery mildew resistance in wheat lines by use of a detached leaf segment technique
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and molecular markers. A preliminary report has been published (Shi et al., 1997).
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1
MATERIALS AND METHODS
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Plant materials
Line #31 is a UK spring wheat line with the pedigree: Wembley*5/Ae. speltoides
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BC5F3 (Miller et al., 1988) and the seeds of Line #31 were obtained from T. E. Miller,
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John Innes Center, Norwich Research Park, Colney, Norwich NR4 7UH, UK. Wembley
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(PI520591) is a UK spring wheat cultivar with pedigree: Hobbit/Sona 227//Sicco, and the
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seeds of Wembley were obtained from Harold E. Bockelman, National Small Grain
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Collection, USDA-ARS, Aberdeen, ID 83210, USA.
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The recurrent parent Wembley, two susceptible cultivars, NK-Coker 68-15 and
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Chancellor, and another twenty differential lines with known Pm genes from Pm1 to
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Pm21 (except Pm10, Pm11, Pm14, Pm15) were used to test response patterns for
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resistance to Bgt isolates. Genes Pm10, Pm11, Pm14, and Pm15 are not effective against
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Bgt (McIntosh et al., 1995) and were not considered here. One series of populations
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involved P1, P2, F1, F2, and BC1F1 were developed from the cross between Line #31 and
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NK-Coker 68-15 from 1994 to 1996 greenhouse seasons. These were used for the
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analysis of the mode of inheritance of resistance to Bgt in Line #31. The populations of
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P1, P2, F1, and F2 derived from the cross between Line #31 and Axminster/8*Cc, which
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contains the single resistance gene Pm1 (McIntosh et al. 1995), were developed for the
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Pm1 allelism test.
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Powdery mildew evaluation
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Powdery mildew evaluations were performed using a detached leaf technique
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(Leath and Heun, 1990). Assessment of reaction was based on a descriptive scale of
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resistant (0-3), intermediate (4-6), and susceptible (7-9) reaction types (Leath and Heun,
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1
1990). Ten differential Bgt isolates were used to test response patterns of resistance to
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powdery mildew in twenty-three wheat materials (Table 1). Three to six Bgt isolates,
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characterized for virulence, were used to test the segregating populations.
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Cosegregation analysis
Cosegregation of resistance to several isolates of Bgt were analyzed by use of the
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detached leaf segment technique in the segregating populations (Schneider et al, 1991).
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Schneider et al. (1991) used this technique to analyze cosegregation of resistance to
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different Bgt isolates in the wheat ‘Normandie’, and found that Normandie contained
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three genes Pm1, Pm2, and pm9, and that pm9 was found to be linked to Pm1 (8.5 cM).
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We utilized this technique as a common laboratory method for analyzing the mode of
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inheritance of resistance to different Bgt isolates. Usually each primary leaf was cut into
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four 2.5 cm length leaf segments, and each segment was inoculated with one Bgt isolate,
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respectively. A segregating population was divided into four groups. Each group was
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inoculated with one isolate, respectively. In that way, the cosegregation of resistance to
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four Bgt isolates were analyzed at the same time on the same leaf. Occasionally each
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primary leaf was cut into five or six leaf segments, and five or six isolates were
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inoculated simultaneously. If the leaf segments per leaf exceeded the isolates, the left
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segments were used as replicates. The NK-Coker 68-15/Line #31 F2 population was
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inoculated with three isolates, 209a2, #8, and Wkin91, in the first test, and with four
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isolates, E314, 137a1, #8, and 153a2, in the second test. The NK-Coker 68-15*2/Line
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#31 BC1F1 and the Line#31/Axminster/8*Cc F2 populations were inoculated with three
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isolates, 209a2, #8, and Wkin91.
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DNA extraction and RAPD analysis
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1
Genomic DNA was extracted from fresh leaves of wheat plants (Doyle and Doyle,
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1990). Random 10-mer primers (kits OP-A through OP-AN) were obtained from Operon
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Technologies Inc. (Alameda CA), and the PCR procedure described by Williams et al.
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(1990) was followed with minor modifications. Each reaction consisted of 2.4 ul reaction
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buffer mix, 1.2 ul dNTPs (2.5mM), 5 ul primer (4 or 5 ng/ul), 0.2 ul Taq polymerase
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(5u/ul), 1.2 ul unacetylated bovine serum albumen (BSA), and 5.0 ul (4 or 5 ng/ul)
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genomic DNA. A total of 41 cycles of PCR amplification were performed using a
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standard RAPD program with denaturation at 92 C for 1 min, annealing at 35 C for 1
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min, and extension at 72 C for 2 min. The reactions were visualized by electrophoesis on
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1.2-1.5% agarose gels in 1X TBE.
RAPD marker screening
RAPD marker screening was approached in two phases. In the first step, 125
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random 10-mer primers were used for screening in the two parents, Line #31 and NK-
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Coker 68-15. In the second step, bulked segregant analysis was used for screening
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RAPD markers by using primers which showed polymorphisms in the two parents.
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A segregating population, NK-Coker 68-15*2/Line #31, was developed and this
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BC1F1 was inoculated with three differential isolates by a detached leaf technique. Four
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phenotypes, R1R2R3, R1R2S3, R1S2S3, and S1S2S3, were observed in this BC1F1
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population. A total of 61 DNA samples extracted from 61 plant leaves, respectively, and
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four bulked DNA samples, R1R2R3 bulked, R1R2S3 bulked, R1S2S3 bulked, and S1S2S3
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bulked, were constructed. The R1R2R3 bulk consisted of 30 DNA samples extracted
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from 30 plants, which were resistant to the three isolates, 209a2, #8, and Wkin 91. The
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R1R2S3 bulk consisted of 10 DNA samples extracted from 10 individuals which were
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1
resistant to two isolates, 209a2 and #8, and susceptible to isolate Wkin 91. The R1S2S3
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bulk consisted of 10 DNA samples extracted from 10 individuals which were resistant to
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isolate 209a2, and susceptible to isolates #8 and Wkin 91. The S1S2S3 bulk consisted of
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nine DNA samples extracted from nine individuals which were susceptible to the three
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isolates 209a2, #8, and Wkin 91. The primers, which revealed polymorphisms in the two
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parents, were used for screening by bulked segregant analysis in the four bulked
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materials, R1R2R3 bulk, R1R2S3 bulk, R1S2S3 bulk, and S1S2S3 bulk, and their parents,
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NK-Coker 68-15 and Line #31.
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Linkage analysis
The 61 DNA samples extracted from 61 individuals in the segregating population
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NK-Coker 68-15*2/Line #31 BC1F1 also were used for linkage analysis of the RAPD
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markers and the resistance alleles in Line #31. Linkage of loci segregating in the BC1F1
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was analyzed based on the maximum likelihood estimator (Allard, 1956; Liu, 1997) and
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calculated by use of a SAS program (SAS Institute Inc., 1990), kindly provided by Dr.
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Ben-Hui Liu, Department of Forestry, North Carolina State University, Raleigh 27695
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(unpublished).
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The recombination frequency was transformed according to the Kosambi function
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using the formula AB = ¼ ln[(1+2r)/(1-2r)] x 100 (Weir, 1996), where AB is the map
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distance (cM). r is estimated recombination frequency. The estimate of standard
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deviation Sr was used the following formula: Sr = % [r (1-r)/N] (Liu, 1997). Goodness-
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of-fit test and independence test were carried out using an P2.
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RESULTS
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Reactions of resistance
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Line #31 was resistant to all tested isolates, and Wembley resistant to six isolates,
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E314, 209a2, 137a1, 101a2, W72-27, and 144, and susceptible to four isolates, #8, E325,
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Wkin91, and 153a2 (Table 1).
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pedigree: Wembley*5/Ae. speltoides BC5F3, the gene(s) for resistance to isolates #8,
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E325, Wkin 91, and 153a2 was introgressed into wheat Line #31 from Ae. speltiodes.
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Inheritance of resistance
Because Line #31 is a UK spring wheat line with
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The three isolates, E314, 144, and 209a2, produced resistant reactions in the NK-
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Coker 68-15/Line #31 F1 progeny, and indicated Line #31 contained dominant resistance
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gene(s). Segregation of resistance to isolate E314, 144, or 209a2 fit a 63R:1S expected
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ratio of three independent dominant genes in the Coker 68-15/Line #31 F2 population,
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and also fit a 7R:1S expected ratio of three independent dominant genes for resistance to
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209a2 in the NK-Coker 68-15*2/Line#31 BC1F1 population (Table 2).
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The reactions of the NK-Coker 68-15/Line #31 F1 progeny were resistant to
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isolates, #8, E325, and 137a1, and showed completely dominance. The segregation of
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resistance to isolate #8, E325, or 137a1 fit a 15R:1S expected ratio for two independent
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dominant genes in the NK-Coker 68-15/Line #31 F2 population, and also fit a 3R:1S
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expected ratio for two independent dominant genes for resistance to #8 in the NK-Coker
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68-15*2/Line#31 BC1F1 population (Table 2).
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The reactions of the NK-Coker 68-15/Line #31 F1 progeny were resistant to
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isolates Wkin 91 and 153a2, and showed completely dominance. Segregation of
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resistance to isolate Wkin 91 or 153a2 fit a 3R:1S expected ratio for one single dominant
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1
gene in the NK-Coker 68-15/Line #31 F2 population, and also fit a 1R:1S expected ratio
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for one single gene for resistance to Wkin91 in the NK-Coker 68-15*2/Line#31 BC1F1
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population (Table 2).
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No plants susceptible to 209a2 were observed in the Line #31/Axminster/8*Cc F2
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population and indicated that an allelic gene existed in the parent lines, Line #31 and
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Aximinster/8*Cc. Because Axminster/8*Cc contained the single resistance gene, Pm1,
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which conditions resistance to 209a2, Line #31 must also carry the gene Pm1. The data
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fit a 15R:1S expected ratio of two dominant genes for resistance to isolate #8, and a
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3R:1S expected ratio of one dominant gene for resistance to isolate Wkin 91 in the Line
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#31/Axminster/8*Cc F2 population (Table 2).
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Cosegregation analysis
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Cosegregation of reactions to three isolates, 209a2, #8, and Wkin 91, fit a
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expected ratio of 48R1R2R3 : 12R1R2S3 : 3R1S2S3 : 1S1S2S3 in the NK-Coker 68-15/Line
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#31 F2 population, and a 4R1R2R3 : 2R1R2S3 : 1R1S2S3 : 1S1S2S3 expected ratio in the
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NK-Coker 68-15 *2/Line #31 BC1F1 population (Table 3). These results indicated that
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the resistance to the three isolates in Line #31 was controlled by three independent
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dominant genes. One gene conferred resistance to the three isolates, another conferred
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resistance to two isolates, 209a2 and #8, and the other conferred resistance only to isolate
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209a2.
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Cosegregation of reactions to the three isolates 209a2, #8, and Wkin 91 also fit a
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expected ratio of 12R1R2R3 : 3R1R2S3 : 1 R1S2S3 : 0 S1S2S3 in the Line #31//Axminster/
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8*Cc F2 population (Table 3).
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Cosegregation of reactions to four isolates, E314, 137a1, #8, and 153a2, of Bgt
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1
also were analyzed in the NK-Coker 68-15/Line #31 F2 population and fit the expected
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ratio of 48R1R2R3R4 : 9R1R2R3S4 : 3R1R2S3 S4 : 3R1S2R3S4 : 1S1S2S3S4 (Table 4). This is
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consistent with the idea that Line #31 contained three independent dominant genes. One
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gene confers resistance to the four isolates, second gene confers resistance to #8 and
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E314, and the third was resistant to 137a1 and E314. Cosegregation was in accordance
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with the 57 R1R2R3 : 3R1R2S3 : 3R1S2 S3 : 1S1S2S3 ratio for reactions to three isolates,
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E314, 137a1, and #8, and the 48R1R2R3 : 12R1R2S3 : 3R1S2 S3 : 1S1S2S3 expected ratio to
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the three isolates, E314, 137a1, and 153a2, and also to the other three isolates, E314, #8,
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and 153a2. These results indicated there were three independent dominant genes in this
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segregating population (Table 4).
Therefore, Line #31 contained three dominant resistance genes. One was Pm12,
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resistant to the all six isolates, E314, 209a2, 137a1, #8, 153a2, and Wkin91; the second
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was Pm1, resistant to E314 and 209a2, and the third was postulated to be Pm2, which
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confers resistance to E314, 209a2, and #8, and susceptible to 137a1, 153a2 and Wkin 91.
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RAPD markers
RAPD markers for Pm12: Ten (out of 125) random 10-mer primers revealed
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polymorphisms in the parent lines, Line #31 and NK-Coker 68-15. Three primers,
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OPAH05, OPAI13, and OPAE12, further revealed polymorphisms by use of bulked
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segregant analysis in the six wheat materials, NK-Coker 68-15, Line #31, R1R2R3 bulk,
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R1R2S3 bulk, R1S2S3 bulk, and S1S2S3 bulk (Fig. 1 using OPAH05). Four RAPD
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markers, OPAH05580, OPAI13490, OPAE12495, and OPAE121350, were present only in
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Line #13 and the R1R2R3 bulk, which contained Pm12, and absent in other seven
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materials (Table 5), and were also observed in individuals of the NK-Coker 68-15*2/Line
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1
#31 BC1F1 population (Fig. 2). The four markers OPAH05580, OPAI13490, OPAE12495,
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and OPAE121350 were linked to Pm12, and the frequency of recombination between
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Pm12 and the four markers was 1.6 " 0.03%, 6.6 " 0.10%, 4.9 " 0.08%, and 3.3 " 0.05%,
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respectively (Table 6). A genetic map was constructed around the gene Pm12 region
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based on data from the NK-Coker 68-15*2/Line #31 BC1F1 population (Fig. 3).
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RAPD markers for Pm1: Two other primers, OPAU17 and OPAI02, revealed
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polymorphisms in the nine wheat materials, Line#31, Wembley, Axminster/8*Cc, NK-
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Coker 68-15, Ulka/8*Cc, R1R2R3 bulk, R1R2S3 bulk, R1S2S3 bulk, and S1S2S3 bulked, and
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the polymorphic bands occurred at about 750-bp and 820-bp. The specific bands were
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present not only in Line#31 and R1R2R3 bulk, but also in the R1R2S3 bulk, R1S2S3 bulk,
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Wembley, and Axminster/8*Cc, but absent in NK-Coker 68-15, Ulka/8*Cc, and S1S2S3
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bulked (Table 5, Fig. 4 using OPAU17). The RAPD marker OPAU17750 was tightly
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linked to Pm1, and the recombination frequency between OPAU17750 and Pm1 is 0.000.
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The 95% confidence interval had bigger range (0 to 9.8 %) based on the data from the
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NK-Coker 68-15*2/Line #31 BC1F1 population, because the samples was not enough big
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accurately to estimate the recombination frequency.
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DICUSSIONS
Cosegregation analysis of resistance alleles
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From the analysis of resistance to the six different isolates, E314, 209a2, #8,
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137a1, 153a2, and Wkin91, of Bgt, individually, it was postulated that resistance in
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wheat Line #31 to isolates E314 and 209a2 was controlled by three independent dominant
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genes; resistance to isolates #8 and 137a1 was controlled by two independent dominant
7
genes; and resistance to isolates Wkin91and 153a2 was controlled by one dominant gene.
8
9
Because the cosegregation of resistance to the three isolates 209a2, #8, and Wkin
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91 of Bgt was in accordance with the expected ratio of 48R1R2R3 : 12R1R2S3 : 3R1S2S3 :
11
1S1S2S3 in the NK-Coker 68-15/Line #31 F2 population, 4R1R2R3 : 2R1R2S3 : 1R1S2S3 :
12
1S1S2S3 in the NK-Coker 68-15*2/Line #31 BC1F1 population, and 12R1R2R3 : 3R1R2S3 :
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1 R1S2S3 : 0 S1S2S3 in the Line #31/Axminster/8*Cc F2 population, it was identified that
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Line #31 contained only three independent dominant genes for resistance to the six
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isolates. One is Pm12, located on the chromosome 6BS/6SS.6SL (Jia et al. 1996),
16
another is Pm1, located on the chromosome 7AL (McIntosh et al. 1995), and the third is a
17
dominant gene and inherited independently from the other two genes, Pm1 and Pm12, in
18
Line #31. This gene was identified to be resistant to E314, 209a2, #8, and susceptible to
19
137a1, Wkin91, and 153a2 by cosegregation analysis and postulated to be Pm2, located
20
on the chromosome 5DS (McIntosh et al. 1995), because the response pattern for this
21
gene was different from those of other Pm genes from Pm1 to Pm21 except Pm2 by gene
22
for gene analysis (Table 1). An allelism test between the Line #31 and a Pm2 line will be
23
helpful to verify that the gene is Pm2.
13
1
R1R2R3 indicates resistance to the three isolates 209a2, #8, and Wkin 91 of Bgt.
2
R1R2S3 signifies resistant to two isolates, 209a2 and #8, and susceptible to isolate Wkin
3
91. R1S2S3 means resistant to isolate 209a2, and susceptible to two isolates, #8 and Wkin
4
91. S1S2S3 was susceptible to the three isolates 209a2, #8, and Wkin 91. In the NK-
5
Coker 68-15/Line #31 F2 population, R1R2R3 plants contained eighteen genotypes, which
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can be grouped into four types: Pm1_ Pm2_ Pm12_, pm1pm1Pm2_ Pm12_, Pm1_
7
pm2pm2Pm12_, and pm1pm1pm2pm2Pm12_, and the expected ratio of the four groups
8
was 4:2:2:1; R1R2S3 plants contained six genotypes, which can be grouped into two
9
types: Pm1_ Pm2_ pm12pm12 and pm1pm1Pm2_ pm12pm12, and the expected ratio of
10
them is 2:1; R1S2S3 plants contained two genotypes Pm1Pm1pm2pm2pm12pm12 and
11
Pm1pm1pm2pm2pm12pm12; S1S2S3 plants contained only one genotype
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pm1pm1pm2pm2 pm12pm12. In the NK-Coker 68-15*2/Line #31 BC1F1 population,
13
R1R2R3 plants contained four genotypes: Pm1pm1Pm2pm2Pm12pm12,
14
pm1pm1Pm2pm2Pm12pm12, Pm1pm1pm2pm2Pm12pm12, and
15
pm1pm1pm2pm2Pm12pm12; R1R2S3 plants contained two genotypes:
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Pm1pm1Pm2pm2pm12pm12 and pm1pm1Pm2pm2pm12pm12; R1S2S3 plants contained
17
one genotype Pm1pm1pm2pm2pm12pm12; and S1S2S3 plants contained one genotype
18
pm1pm1pm2pm2pm12pm12. From the four types of phenotypes, all R1R2R3 plants
19
contained the Pm12 gene; All R1R2S3 plants contained the Pm2 gene; All R1S2S3 plants
20
contained the Pm1 gene; and all S1S2S3 plants contained no resistance gene.
21
expected ratio of cosegragation for resistance to the three isolates, 209a2, #8, and Wkin
22
91, was in accordance with the ratio of 48R1R2R3 : 12R1R2S3 : 3R1S2S3 : 1S1S2S3 in the
23
NK-Coker 68-15/Line #31 F2 population, postulated from (3Pm1 _ : 1pm1pm1) (3Pm2 _ :
The
14
1
1pm2pm2) (3Pm12 _ : 1 pm12pm12) = [(3Pm1 _ : 1pm1pm1) (3Pm2 _ :
2
1pm2pm2)(3Pm12 _)] : [9 Pm1 _ Pm2 _ pm12 pm12 : 3 pm1pm1Pm2_ pm12pm12] : [3
3
Pm1 _ pm2pm2pm12pm12] : [1 pm1pm1pm2pm2pm12pm12] = 48R1R2R3 : 12R1R2S3 :
4
3R1S2S3 : 1S1S2S3. The expected ratio for resistance to the three isolates, 209a2, #8, and
5
Wkin 91, is 4R1R2R3 : 2R1R2S3 : 1R1S2S3 : 1S1S2S3 in the NK-Coker 68-15*2/Line #31
6
BC1F1 population, postulated from (1Pm1pm1 : 1pm1pm1) (1Pm2pm2 : 1pm2pm2)
7
(1Pm12pm12 : 1pm12pm12) = [1Pm12pm12(1Pm1pm1: 1pm1pm1)(1Pm2pm2:pm2pm2)]
8
: [2_pm1Pm2pm2pm12pm12]: [1Pm1pm1pm2pm2pm12pm12] :
9
[1pm1pm1pm2pm2pm12pm12] = 4R1R2R3 : 2R1R2S3 : 1R1S2S3 : 1S1S2S.
10
With the same reasons, the expected ratios of cosegregation for resistance to the
11
different isolates of Bgt were postulated in the NK-Coker 68-15/Line #31 F2 population
12
(Table 3 and 4).
13
14
RAPD markers analysis
The RAPD marker, OPU17750, which was tightly linked to Pm1 (2.2  1.07 cM),
15
was found to be present in the wheat Line #31 (Chapter 3). In this research, the four
16
DNA samples were bulked from the NK-Coker 68-15 *2/Line #31 BC1F1 population.
17
The R1R2R3 bulked consisted of 30 R1R2R3 phenotype BC1F1 plants, among which half
18
plants contained Pm1 gene; the R1R2S3 bulked consisted of 10 R1R2S3 phenotype BC1F1
19
plants, of which about five plants contained the Pm1 gene; the R1S2S3 bulk consisted of
20
10 R1S2S3 phenotype plants, among which all plants contained the Pm1 gene. Eight
21
possible phenotypes are R1R2R3M, R1R2R3m, R1R2S3M, R1R2S3m, R1S2S3M, R1S2S3m,
22
S1S2S3M, and S1S2S3m between the resistance to three isolates and an given marker in
23
this BC1F1 population. It is assured that the recombination frequency between a
15
1
resistance gene and a marker is r, the expected ratio is 2(1+r) : 2(1-r) : (1+r) : (1-r) : (1-r)
2
: r : r : (1-r) corresponding the eight phenotypes. Since the observed number was 18, 17,
3
9, 8, 10, 0, 0, and 9 corresponding the eight phenotypes, the recombination frequency r =
4
0.000 was calculated based on the data from the NK-Coker 68-15*2/Line #31 BC1F1
5
population by use of the maximum likelihood method.
6
All 10 R1S2S3 phenotypic individuals also revealed the presence of the marker
7
OPU17750, and in all nine S1S2S3 phenotypic individuals, the marker was absent. This
8
indicated that no crossovers took place between the marker and Pm1 and that OPU17750
9
was tightly linked to Pm1.
10
In this research, if only one isolate was used, the linkage between Pm1 and the
11
RAPD marker could not be determined in the NK-Coker 68-15*2/Line #31 BC1F1
12
population or in the NK-Coker 68-15/Line #31 F2 population, because all of our
13
characterized isolates, which are avirulent to Pm1, are also avirulent to Pm12. Therefore,
14
cosegregation of resistance to different isolates by use of different Bgt isolates on separate
15
segments of the same leaf is a useful method for identifying more than one gene and
16
analyzing molecular markers linked to different genes in one resistant line.
17
Prior to this research, the presence of Pm1 gene in the wheat Line #31 was
18
unknown (Miller et al, 1988). Because the two RAPD markers OPAU17750 and
19
OPAI02820 were found to be present in the wheat Line #31, a cross between the wheat
20
Line #31 and Axminster/8*Cc was made, and the Line #31/Axminster/8*Cc F2
21
population was developed. An allelism test indicated the wheat Line #31 also carried
22
gene Pm1.
23
The four RAPD markers, OPAH05580, OPAI13490, OPAE12495, and OPAE121350,
16
1
were tightly linked gene Pm12, and can be used for marker-assisted selection for Pm12.
2
Because Pm12 is located on chromosome 6B-6SS.6SL, the four RAPD markers can be
3
used in the genetic mapping of this chromosome.
4
Resistance evaluation
5
Wembley was resistant to isolates E314, 209a2, 137a1, 101a1, W72-27, and 144,
6
and susceptible to #8, E325, Wkin91 and 153a2, and postulated to contain Pm1 by host-
7
pathogen interaction and linked RAPD marker analysis. The RAPD marker, OPAU17750,
8
tightly linked to Pm1 also was present in Wembley. This indicated Wembley contained
9
Pm1. Therefore, the gene Pm1 in Line #31 was derived from its recurrent parent
10
Wembley. The gene Pm12 in Line #31 was transferred from Ae. speltoides. The other
11
gene Pm2 could also have been derived from the wild relative, because Wembley was
12
susceptible to isolates #8 and E325, and didn’t contain gene Pm2.
13
Miller et al (1988) reported Line #31 carried a single dominant gene Pm12 and
14
was highly resistant to all Bgt isolates in Europe. Reaction response patterns for
15
resistance to wheat powdery mildew were tested with 30 to 80 Bgt isolates in 26
16
differential lines with Pm genes from Pm1 to Pm21, and only Pm12, Pm13, Pm20, and
17
Pm21 lines were resistant to all tested isolates (data are not shown). A total of 520
18
isolates collected from seventeen States of USA were tested for virulence genes using
19
fifteen lines with known Pm resistance genes from Pm1 to Pm17, and no isolate was
20
virulent to the gene Pm12 (Niewoehner and Leath, 1997). Therefore Pm12 remains an
21
excellent gene for resistance to wheat powdery mildew.
17
REFERENCES
Allard, R.W. 1956. Formulas and tables to facilitate the calculation of recombination
values in heredity. Hilgardia 24:235-279.
Doyle, J. J. and J. L. Doyle. 1987. Isolation of plant DNA from fresh tissue. Focus
12:13-15.
Jia, J., K. M. Devos, S. Chao, T. E. Miller, S. M. Reader, and M. D. Gale. 1996. RFLPbased maps of the homoeologous group-6 chromosomes of wheat and their
application in the tagging of Pm12, a powdery mildew resistance gene transferred
from Aegilops speltoides to wheat. Theor. Appl. Genet. 92:559-565.
Leath, S., and M. Heun. 1990. Identification of powdery mildew resistance genes in
cultivars of soft red winter wheat. Plant Dis. 74:747-752.
Liu, B. 1997. Statistical Genomics --Linkage, Mapping, and QTL Analysis. CIC Press,
Boca Raton, Florida.
McIntosh, R.A., G. E. Hart, and M. D. Gale. 1995. Catalogue of gene symbols for wheat.
Pp. 1333-1500 In: Z. S. Li and Z. Y. Xin eds. Proceedings 8th International Wheat
genetics symposium, Beijing, China, 1993.
Michelmore, R. 1995. Molecular approaches to manipulation of disease resistance
genes. Annu. Rev. Phytopathol. 15:393-427.
Miller, T. E., S. M. Reader, C. C. Ainsworth, and R. W. Summers. 1988. The
introduction of a major gene for resistance to powdery mildew of wheat, Erysiphe
graminis f. sp. tritici, from Aegilops speltoides into wheat, Triticum. In: M.L.
Jorna, L.A.J. Slootmaker (eds) Cereal breeding related to integrated cereal
production. Pudoc, Netherlands, pp 179-183.
18
Niewoehner, A. S., and S. Leath. 1997. Determing virulence frequencies of Blumeria
graminis f. sp. tritici on soft red winter wheat. Plant Dis. (in press).
SAS Institute Inc. 1990. SAS/ATAT Userd's Guide, Version 6, Fourth Edition. Cary NC,
USA.
Shi, A. N., S. Leath, and J. P. Murphy. 1997. Identification of RAPD markers linked to
two major genes for powdery mildew resistance in Pm12 wheat line.
Phytopathology 87:S89
Stuber, C. W. 1992. Biochemical and molecular markers in plant breeding, pp. 37-61. In
J. Janick (ed.), Plant Breeding Reviews, Vol. 9. John Wiley & Sons, Inc., New
York.
Weir, B. S. 1996. Genetic Data Analysis II. Methods for Discrete Population Genetic
Data. Sinauer Associates, Inc. Publisher, Sunderland, MA.
Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski, and S.V. Tingey. 1990. DNA
polymorphisms amplified by arbitrary primers are useful as genetic markers.
Nucleic Acid Res. 18: 6531-6535.
19
Table 1. Reactions of twenty-three wheat lines for powdery mildew resistance after inoculation with ten isolates
of Blumeria graminis f. sp. tritici.
_________________________________________________________________________________________
Line
Gene
Isolate of Blumeria graminis f. sp. tritici
E314 209a2 137a1 101a1 #8 E325 Wkin91 153a2 W72-27 144
_________________________________________________________________________________________
Chancellor
None
S
S
S
S
S
S
S
S
S
S
NK-Coker 68-15
None
S
S
S
S
S
S
S
S
S
S
Axminster/8*Cc
Pm1
R
R
R
R
S
S
S
S
R
R
Ulka/8*Cc
Pm2
R
R
S
S
R
R
S
S
R,I
R
Asosan/8*Cc
Pm3a
R
S
S
S
R
R
S
S
S,I
S,I
Chul/8*Cc
Pm3b
R
R
I
S
R
R
R
R
R
S
Sonora/8*Cc
Pm3c
R
R
S
S
S
S
S
R
S,I
S
Khapli/8*Cc
Pm4a
S
S
S
S
S
S
R
R
S
S
Ronos
Pm4b
S
S
R
R
S
S
R
R
R
R
Kormoran
Pm5
S
S
S
S
S
S
S
S
S
S
Coker 747
Pm6
R
S
S
S
S,I
I
S
S
S
S
Transec
Pm7
S
S
S
S
S
S
S
S
S
S
Kavkaz
Pm8
S
R
R
R
S
S
R
R
R
R
Normandie
Pm9+1+2
S
R
R
R
R
R
R,I
I
I
R
Chinese Spring
Pm13
R
R
R
R
R
R,I
R
R
R
R
BRG 3N/76
Pm16
R
R
R
R
R
R,I
R,I
S,I
R,I
R
Amigo
Pm17
S
R
I
R,I
S
I
R
R,I
R,I
R
Weihenstephan M1N Pm18
R
R,I
R
R
R
R,I
R,I
R
R
R
D85350
Pm19
R,I
I
S,I
S
I
I
S,I
S,I
S
S,I
TAM104/Thatcher Pm20
R
R,I
R,I
R,I
R
R
R
R,I
R,I
R
PM941181
Pm21
R
R
R
R
R
R
R
R
R
R
Wembley
Pm1
R
R
R
R
S
S
S
S
R
R
Line #31
Pm12+1+2
R
R
R
R
R
R
R
R
R
R
_________________________________________________________________________________________
† R = resistant, I = intermediate, and S = susceptible.
20
Table 2. Segregation of resistance to Blumeria graminis f. sp. tritici isolates in three segregating wheat populations
____________________________________________________________________________________________________
F1
No. of observed
Cross
Isolate
Resistant Susceptible
Exp.
P2
P value
___________________________________________________________________________________________________
NK-Coker 68-15/Line #31 F2
E314
R
198
6
63:1
2.521
0.10-0.25
209a2
R
393
5
63:1
0.243
0.50-0.75
137a1
R
187
17
15:1
1.511
0.10-0.25
#8
R
552
42
15:1
0.683
0.25-0.50
Wkin91
R
300
98
3:1
0.030
0.75-0.90
153a2
R
144
60
3:1
2.118
0.10-0.25
NK-Coker 68-15*2/Line #31 BC1F1 209a2
R
81
9
7:1
0.514
0.75-0.90
#8
R
71
19
3:1
0.726
0.25-0.50
Wkin 91
R
50
40
1:1
1.111
0.25-0.50
209a2
R
289
0
1:0
0.000
1.00
#8
R
276
13
15:1
1.513
0.25-0.50
Line #31/Axminster/8*Cc F2
Wkin 91
R
224
65
3:1
0.970
0.25-0.50
_____________________________________________________________________________________________________
21
Table 3. Cosegregation of resistance to three isolates, 209a2, #8, and Wkin91, of Bgt in three segregating wheat populations
______________________________________________________________________________________________________
Isolate
F2 or BC1F1 phenotype
Cross
1
2
3
R1R2R3 R1R2S3 R1S2S3 S1S2S3 Expected ratio
P2
______________________________________________________________________________________________________
NK-Coker 68-15/Line #31 F2
209a2
#8
Wkin91
300
75
18
5
48:12:3:1
0.271
NK-Coker 68-15*2/Line #31 BC1F1 209a2
#8
Wkin91
50
21
10
9
4:2:1:1
1.244
Line #31/Axminster/8*Cc F2
#8
Wkin91
224
52
13
-
12:3:1
1.750
209a2
_____________________________________________________________________________________________________
† R1R2R3 = resistant to the three isolates 209a2, #8, and Wkin 91, R1R2S3 = resistant to two isolates 209a2 and #8, and
susceptible to isolate Wkin 91, R1S2S3 = resistant to isolate 209a2, and susceptible to two isolates #8 and Wkin 91, and
S1S2S3
= susceptible to the three isolates 209a2, #8, and Wkin 91.
22
Table 4. Cosegregation of resistance to four isolates E314, 137a1, #8, and 153a2 of Bgt in the NK-Coker 68-15/Lin 31 F2
wheat population
_____________________________________________________________________________________________________
Isolate
F2 phenotype
1
2
3
R1 R2 R3
R1 R2 S 3
R1 S 2 S 3
S1S2S3
Expected ratio
P2
P value
____________________________________________________________________________________________________
E314
137a1 #8
177
10
11
6
57:3:3:1
2.839
0.25-0.50
E314
137a1 153a2
144
43
11
6
48:12:3:1
3.817
0.10-0.25
E314 #8
153a2
144
44
10
6
48:12:3:1
3.895 0.10-0.25
---------------------------------------------------------------------------------------------------------------------------------------------------Isolate
F2 phenotype
1
2
3
4
R1R2R3R4 R1R2R3S4 R1R2S3 S4 R1S2R3S4 S1S2S3S4 Expected ratio P2
P value
E314
137a1 #8
153a2
144
33
10
11
6
48:9:3:3:1
3.895
0.25-0.50
23
Table 5. RAPD markers from bulked segregant analysis in nine wheat materials
_________________________________________________________________________________________
Line
OPAE12495 OPAE121350 OPAI13490
OPAH05580 OPU17750
OPAI02800
_________________________________________________________________________________________
Coker 68-15
Line #31
+
+
+
+
+
+
R1R2R3 bulked
+
+
+
+
+
+
R1R2S3 bulked
+
+
R1S2S3 bulked
+
+
S1S2S3 bulked
Axminster/8*Cc
+
+
Wembley
+
+
Ulka/8*Cc
_________________________________________________________________________________________
† '+' = the band present, '-' = the band absent.
Table 6. Cosegregation of Pm12 for resistance to isolate Wkin 91 of Bgt and RAPD markers in the
NK-Coker 68-15*2/Line #31 BC1F1 wheat population.
_________________________________________________________________________________
BC1F1 phenotype
P2A P2B P2AB
Recombination
A
B
RAMB RAmB SAMB SAmB (1:1) (1:1) (1:1:1:1)
frequency (%)
_________________________________________________________________________________
Pm12 OPAE12495 31
1
2
27
0.148 0.410 50.147**
4.9  0.08
Pm12 OPAE121350 32
0
2
27
0.148 0.803 54.213**
3.3  0.05
Pm12 OPAI13490
31
1
3
26
0.148 0.803 47.000**
6.6  0.10
Pm12 OPAH05580 32
0
1
28
0.148 0.410 57.623**
1.6  0.03
_________________________________________________________________________________
† ** is significant at P=0.01.
‡ R = resistant, S = susceptible, M = the marker present, and m = the marker absent.
24
Fig. 1. Amplification pattern from RAPD marker
OPH05580 for Pm12 in six wheat materials:
1. NK-Coker 68-15 (None),
2. Line #31 (Pm1, Pm2, Pm12),
3. R1R2R3 bulked (Pm1, Pm2, Pm12),
4. R1R2S3 bulked (Pm1, Pm2),
5. R1S2S3 bulked (Pm1),
6. S1S2S3 bulked (None).
580bp
Fig. 2. Amplification pattern from OPAH05580 RAPD fragments in the NK-Coker 6815*2/Line #31 BC1F1 population. Lane R is the resistant individual and lane S is the
susceptible individual in the BC1F1 population. Lane M is a 1-kb molecular-weight
marker.
25
1.6
OPAI13490
OPAE12495
3.3
1.6
OPAH05580
Pm12
3.3
OPAE121350
Fig. 3. A genetic map of the region carrying Pm12 constructed
from the NK-Coker 68-15*2/Line #31 BC1F1 population.
1 2
3
4
5
6 7 8
Fig. 4. Amplification pattern of DNA detecting RAPD
marker OPU17750 for Pm1 in seven wheat materials:
1. NK-Coker 68-15 (None),
2. Wembley (Pm1),
3. Line #31 (Pm1, Pm2, Pm12),
4. R1R2R3 bulked (Pm1, Pm2, Pm12),
5. R1R2S3 bulked (Pm1, Pm2),
6. R1S2S3 bulked (Pm1),
7. S1S2S3 bulked (None),
8. Axminster/8*Cc (Pm1).