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a
a Pioneer Hi-Bred International, Inc., 7300 NW 62nd Avenue, P.O. Box 1004, Johnston, IA 50131-1004
b Dep. of Crop Science, North Carolina State Univ., Raleigh, NC 27695
c Dep. of Plant Pathology, North Carolina State Univ., Raleigh, NC 27695
d USDA-ARS, and Dep. of Plant Pathology, North Carolina State Univ., Raleigh, NC 27695
* Corresponding author (njpm{at}unity.ncsu.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: AFLP, amplified fragment length polymorphism • SSR, simple sequence repeat, or microsatellite
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Host plant resistance is the most cost effective means for powdery mildew control. Monogenic resistance is mainly via a hypersensitive foliar reaction involving major Pm genes in a gene-for-gene interaction (Bennett, 1984; Chen and Chelkowski, 1999; Hsam and Zeller, 2002). Thirty major gene loci have been identified to date in common wheat (McIntosh et al., 1998; Rong et al., 2000; Järve et al., 2000; Peusha et al., 2000; Zeller et al., 2002; Liu et al., 2002; Hsam et al., 2003; Singrun et al., 2003). Horizontal or quantitative resistance has been identified (Shaner, 1973; Griffey and Das, 1994; Chantret et al., 2001; Liu et al., 2001) and adult plants with this form of resistance exhibit a decrease in disease intensity compared with the fully susceptible lines.
Common sources of Pm genes are species within the primary, secondary, and tertiary gene pools of wheat (Hsam and Zeller, 2002). A program of interspecific hybridization between powdery mildew resistant diploid and tetraploid relatives and the soft red winter wheat cultivar Saluda (Starling et al., 1986) was initiated in 1986 by the small grains breeding and pathology projects at North Carolina State University. To date 11 germplasm lines have been released (Murphy et al., 1998, 1999a, 1999b, 2002; Navarro et al., 2000).
Selection of lines containing major Pm genes in breeding nurseries in the mid-Atlantic states is facilitated by annual powdery mildew epidemics, but selection of lines containing pyramids of Pm genes necessitates the use of molecular markers linked to resistance genes. There have been numerous demonstrations of the utility of molecular markers in wheat improvement, including the tagging of major Pm genes and quantitative trait loci (QTL) associated with horizontal resistance (reviewed by Huang and Roder, 2004). The multi-allelic Pm1 locus on chromosome 7AL has been studied using restriction fragment length polymorphism (RFLP) (Ma et al., 1994; Hartl et al., 1995, 1999), randomly amplified polymorphic DNA (RAPD) (Hu et al., 1997), AFLP (Hartl et al., 1999), and SSR marker systems (Neu et al., 2002; Singrun et al., 2003).
Knowledge of the inheritance of resistance to powdery mildew and the linkage between the resistance genes and molecular markers in the recently released North Carolina germplasms would be advantageous for marker assisted selection in cultivar development. The objectives of this study were to determine the inheritance, chromosomal location, and linkage with molecular markers of genes for resistance to powdery mildew in two of these germplasm lines, NC96BGTA4 and NC99BGTAG11.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Greenhouse Evaluation of Inheritance of Resistance to Powdery Mildew
Ninety-nine F2:3 lines from each of the two populations were evaluated for reaction to powdery mildew in separate experiments during 2001 and 2002. An experimental unit was two 10-cm pots each planted with five F2:3 seeds of each line. The experimental design was a completely randomized design with a single replication. Two pots containing the relevant parental germplasm line and Saluda were included at 10-pot intervals as controls. Each control pot contained two plants. Seeds of parental germplasm lines and Saluda were derived from selfed progenies of the plants used in crosses to develop the two populations. The seeds were planted in a mixture of Metro-Mix 200 (Scotts-Sierra Horticultural Products Co., Marysville, OH), soil, and sand in a 50:40:10 ratio. Three grams of a slow release 14–14–14 (N–P–K) fertilizer was mixed with the potting medium in each pot. The temperature was maintained at 24°C (day) and 20°C (night). Plants were grown under a combination of plentiful natural light supplemented with artificial high intensity 1000-W discharge lights.
Plants were inoculated at Feekes growth stage 2 to 3 (Large, 1954) by gently shaking conidia from leaves of infected Saluda plants onto leaves of F2:3 lines and parental plants. The inoculum source was field grown Saluda plants dug at the Cunningham Research and Education Center, Kinston, NC, in April 2001. The inoculum was maintained through the year on Saluda plants in the greenhouse and under laboratory conditions on detached leaves according to the method of Leath and Heun (1990). Disease evaluations were conducted when all Saluda control plants showed abundant signs and symptoms of powdery mildew infection. The disease severity evaluation was on a scale from 0 to 9 as described by Leath and Heun (1990) where: 0 = immune, no visible signs of infection; 1 to 3 = resistant, increasing from (i) flecks, no necrosis to (ii) necrosis, to (iii) chlorosis, while the amount of mycelium went from none to a detectable amount; 4 to 6 = intermediate, chlorotic areas decreasing in amount while mycelium and conidia production increased from slight to moderate; 7 to 9 = susceptible, increasing amount, size, and density of mycelium and conidia to a fully compatible reaction. Disease reactions and intensities in F2:3 progeny similar to the parental germplasm line were classified as "resistant" and those similar to Saluda were classified as "susceptible."
Field Evaluations of Inheritance of Resistance to Powdery Mildew
We planted 189 to 194 F2:3 lines from each of the two populations at Kinston, NC, in October 2001. All F2:3 lines evaluated in the greenhouse tests were evaluated in the field tests. The experimental design was a randomized complete block with two replications. An experimental unit was a 1.2-m row sown with 20 to 60 seeds of each line. Rows were spaced 30.5 cm apart. Parental germplasm lines and Saluda were included as controls every 40 plots. In addition, each replication contained the cultivar Chancellor and 12 isolines with previously identified Pm genes backcrossed into Chancellor. The donor source and the major gene in each isoline were as follows: Axminster (Pm1a), Ulka (Pm2), Asosan (Pm3a), Chul (Pm3b), Sonora (Pm3c), Michigan Amber (Pm3f), Yuma (Pm4), Hope (Pm5a), Coker 747 (Pm6), Transec (Pm7), Federation/Kavkaz (Pm8), and Amigo (Pm17). Chancellor contains no Pm genes that are effective against wheat powdery mildew, but it contains Pm10 and Pm15 that are effective against wheatgrass powdery mildew (caused by Blumeria graminis DC f. sp. agropyri) (Briggle, 1969; Tosa and Tada, 1990). A 1.2-m border of Saluda surrounded the experiment. Irrigation, fertilization, and other agronomic practices, excluding fungicide application, followed standard management practices for North Carolina (Weisz, 2000). Homogeneous resistant and susceptible lines in both populations were harvested in June 2002. F2:4 seed of a random sample of the homogeneous resistant and susceptible lines, NCA4, NCAG11, Saluda, Chancellor, and the 12 isolines were reevaluated at Kinston, NC, in the 2002–2003 season using the same protocols.
Disease reaction evaluations were initiated at the end of March 2002 and 2003 when all Saluda plots showed uniform powdery mildew infection. Plants were between Feekes growth stage 9 and 10.1. Flag minus 2 leaves were evaluated using the modified 0 to 9 scale of Leath and Heun (1990). Disease reactions were recorded on 12 to 24 random plants in each row. A sample size of 11 is required to identify a single recessive plant with a P = 0.95 in a population segregating with a 3:1 ratio (Sedcole, 1977). Lines observed as segregating for resistance in one replication and homogenous for resistance in the other were classified as segregating.
Chi-square tests were conducted to evaluate the goodness of fit between observed and expected segregation ratios in the two populations (Snedecor and Cochran, 1956). The GLM procedure of the SAS software (SAS Institute, 1999) was utilized in the analysis of field data on parental and isogenic lines. The least significant difference (LSD) for comparison between germplasm and isogenic lines was computed as LSD = t[MSE(g–1 + i–1)]1/2, where MSE was the estimated error mean square for testing the significance of variance among lines, g and i were the number of replicates per germplasm or isoline, respectively, and t was the t value at the P = 0.05 probability level for the number of degrees of freedom associated with MSE.
Field Evaluation of Resistance to Powdery Mildew among Parental Lines
NCA4, NCAG11, and Saluda were planted in two replicate randomized complete block experiments between 20 October and 15 November in 2000, 2001, 2002, and 2003 at Kinston. Plots were 5.1 m2 with seven rows spaced 18 cm apart. Irrigation, fertilization, and other agronomic practices, excluding fungicide application, followed standard management practices for North Carolina (Weisz, 2000). Powdery mildew severity was evaluated on a whole-plot basis once each spring between Feekes growth stage 8 and 10.1. Assessments were based on the extent and position of lesions in the canopy. A 0 to 9 scale was utilized where: 0 = no detectable lesions; 1 to 3 = lesions ranging from barely detectable to 1% coverage of leaf area in lower one-third of canopy; 4 to 6 = lesions ranging from barely detectable to 5% coverage of leaf area in middle third of canopy; and 7 to 9 = lesions covering from 1% of the flag minus 1 leaf to 10% or more of flag leaf.
DNA Extraction
DNA was extracted from young leaf tissue of greenhouse-grown parental lines, 115 F2 plants from the Saluda x NCA4 population and 127 F2 plants from the Saluda x NCAG11 population. The F2 plants were a random subset of those utilized to produce F2:3 lines for field evaluations of powdery mildew resistance, but included all F2 plants utilized to produce F2:3 lines for greenhouse evaluations. In addition, DNA was extracted from one F4 plant in each of 23 F2:4 lines homozygous for resistance or susceptibility in the Saluda x NCAG11 population and from 10 F5 individuals in each of three F4:5 lines homozygous for resistance in the Saluda x NCA4 population. The DNeasy Plant Mini Kit (Qiagen, Valencia, CA) was used following the manufacturer's instructions, and DNA concentrations were adjusted to 200 ng per 12 µL of reaction mix.
Amplified Fragment Length Polymorphism Analysis
Restriction and ligation were performed according to the protocol supplied by Life Technologies (Gaithersburg, MD) with the Core Reagent Kit. Preamplifications were performed with EcoRI + A and MseI + C primers (Life Technologies). Polymerase chain reaction (PCR) conditions for preamplifications were: 94°C for 30 s, 60°C for 30 s, and 72°C for 60 s (27 cycles). The selective amplifications were done with EcoRI + 3 and MseI + 3 primer combinations. Sequences of EcoRI primers (LI-COR, Lincoln, NE) were 5'-GACTGCGTACCAATTCNNN-3' and MseI primers (Sigma-Aldrich, Milwaukee, WI) were 5'-GATGAGTCCTGAGTAANNN-3'. The PCR conditions for selective amplifications were: 94°C for 30 s, 65°C for 30 s, and 72°C for 60 s (12 cycles); 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s (22 cycles). The electrophoresis was conducted in LI-COR sequencers, Models 4000 and 4200L, on 8% polyacrylamide denaturing gels under 48°C, 42 W, 35 mA, and 1500 V for approximately 3 h. Electronic images of the gels were analyzed by AFLP Quantar 1.09 software (KeyGene Products, 2000).
Screening for polymorphisms between Saluda and NCA4 or NCAG11 utilized 88 primer combinations. Subsequently, the selected primer combinations were used to compare bulks containing DNA of 10 homozygous resistant F2 individuals with bulks containing DNA of 10 homozygous susceptible F2 individuals. The inheritance of polymorphisms identified through bulked segregant analyses was investigated utilizing 127 F2 individuals in the Saluda x NCAG11 population. The AFLP marker nomenclature followed the protocol outlined at the GrainGenes website (http://wheat.pw.usda.gov/ggpages/keygeneAFLPs.html; verified 7 Mar. 2005).
Simple Sequence Repeat Analysis
Microsatellite primer sequences were obtained from Röder et al. (1998) and the GrainGenes database (http://wheat.pw.usda.gov/; verified 7 Mar. 2005). The SSR loci selected for screening for polymorphisms between Saluda and NCA4 or NCAG11 were evenly distributed across the A and B genomes. All primers were obtained from MWG Biotech (High Point, NC). Forward primers were modified to incorporate the M13 primer sequence (5'-CACGACGTTGTAAAACGAC-3') for the purpose of universal fluorescent labeling (Schuelke, 2000; Rampling et al., 2001). M13 primer was labeled with IRDye800 or IRDye700.
The PCR reactions contained 1x PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs (Promega, Madison, WI), 0.15 pM forward primer, 0.75 pM reverse primer, 0.75 pM M13 labeled primer, 0.2 µL BSA (10 µg µL–1; NEB, Beverly, MA), 0.75 U Taq polymerase (CLP, San Diego, CA), and 50 ng genomic DNA in a 10-µL total volume. Cycling was completed using the touchdown program as written by Rampling et al. (2001).
Reactions were diluted 1:1 with 95% formamide buffer [19 mL formamide (Fisher Scientific, Hampton, NH)], 1.0 mL 0.5 M EDTA (Fisher Scientific), pH 8.0, 0.16 g bromophenol blue (USB, Cleveland, OH), denatured for 3 min at 90°C, and placed immediately on ice. Samples were loaded onto 6.5% denaturing polyacrylamide gels and run on LI-COR sequencers for 2.5 h at 48°C, 42 W, 35 mA, and 1500 V. Gels were scored using AFLP Quantar 1.09 software (KeyGene Products, 2000).
A total of 188 SSR loci were examined for polymorphism between the parents in both populations. Subsequently, the selected primer combinations were used for bulked segregant analysis of DNA samples prepared as described from resistant and susceptible individuals for each population. Forty-eight primer combinations were tested in the Saluda x NCA4 population, and 50 primer combinations were tested in the Saluda x NCAG11 population. The inheritance of polymorphisms identified through bulked segregant analyses was investigated utilizing 115 F2 individuals in the Saluda x NCA4 population and 105 F2 individuals in the Saluda x NCAG11 population.
Linkage Analysis
Linkage relationships between marker loci and resistance genes were determined by Mapmaker/Exp (Version 3.0 b) (Lincoln et al., 1993). Map distances were calculated using the Kosambi function to correct for crossover interference in estimation of recombination fractions. The decimal logarithm of odds (LOD) ratio was set to 3.0. Loci were ordered on the chromosome using the "sequence" and "compare" commands.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Field
Saluda had a mean disease rating of 7.7 ± 0.67 and a range of 7.0 to 9.0 (Table 1). NCA4 was immune with a mean of 0.0. Forty-one F2:3 lines were homozygous resistant (Table 2). One-hundred-eight F2:3 lines were segregating. Forty-five F2:3 lines were homozygous susceptible, with a mean of 7.1 ± 0.64 and a range of 6.0 to 8.0. The chi-square test value for the expected 1:2:1 ratio was 2.66 (P = 0.26), indicating that resistance evaluated in the field segregated as a monogenic trait.
NCA4 and the resistant progenies had notably different disease reactions in the greenhouse versus the field environment, while Saluda and the susceptible progenies had similar reactions in both environments. In the greenhouse, NCA4 and the resistant progenies exhibited an intermediate response with chlorosis and some mycelium production. NCA4 and the resistant progenies had an immune reaction in the field in 2002. The different disease ratings in the two environments may have reflected differences due to stage of plant development (Shaner, 1973; Griffey and Das, 1994). It is also possible that the powdery mildew population in the greenhouse had a more complex pathogenicity profile, such as multiple avr genes, so the resistance gene in NCA4 was very effective against some powdery mildew isolates and less effective against others. Although differences in level of resistance expression were observed in the two environments, both data sets indicated that NCA4 contained a single major resistance gene.
Cross 2 (Saluda x NCAG11)
Greenhouse
Saluda had a mean disease rating of 7.5 ± 0.51 and a range of 7.0 to 8.0 (Table 1). NCAG11 was immune. Twenty-six F2:3 lines were homozygous resistant with a mean of 0.0 (Table 2). Forty-one F2:3 lines were segregating. Thirty-two lines were homozygous susceptible. The chi-square test value for the expected 1:2:1 ratio was 3.65 (P = 0.16), indicating that resistance evaluated in the greenhouse segregated as a monogenic trait.
Field
Saluda had a mean disease rating of 7.1 ± 0.57 and a range of 6.0 to 8.0 (Table 1). NCAG11 was immune. Fifty-two F2:3 lines were homozygous resistant with a mean of 0.0 (Table 2). Eighty-one F2:3 lines were segregating. Fifty-six F2:3 lines were homozygous susceptible with a mean of 7.2 ± 0.53 and a range of 5.0 to 8.0. The chi-square test value for the expected 1:2:1 ratio was 4.03 (P = 0.13), indicating that resistance evaluated in the field segregated as a monogenic trait.
Identification of Molecular Markers Linked to the Resistance Gene in NCA4
Fifty-five AFLP primer combinations generated 167 polymorphisms between Saluda and NCA4, but none were considered viable markers for the resistance gene in NCA4 following bulked segregant analysis. The NCA4 x Saluda population was not subjected to further AFLP investigation.
Polymorphisms between Saluda and NCA4 were detected using bulked segregate analysis at the Xwmc525, Xbarc292, and Xgwm4 SSR loci (Fig. 1). Xwmc525 was the only locus displaying codominant gene action and the stutter observed was consistent with the description of this locus in GrainGenes. A 211 bp fragment was observed at the Xwmc525 locus in NCA4 and a 230 bp fragment was observed in Saluda. The large allele separation at this locus allowed for consistent scoring of this locus despite the stutter. A 219 bp fragment was observed in Saluda at Xbarc292. A 253 bp fragment was observed in Saluda at Xgwm4. The F2 segregation pattern at the Xwmc525 locus followed the expected 1:2:1 ratio of a codominant marker while the segregation patterns at the Xgwm4 and Xbarc292 loci followed the expected 3:1 ratio of a single dominant marker (Table 3). The inheritance of powdery mildew resistance in the F2:3 lines derived from the random F2 plants that underwent SSR analyses followed the expected 1:2:1 ratio (data not shown).
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Identification of Molecular Markers Linked to the Resistance Gene in NCAG11
Fifty-two AFLP primer combinations generated 138 polymorphisms between Saluda and NCAG11 and two AFLPs, XE36M55-126 and XE38M54-196, were selected for further study based on bulked segregant analysis (Fig. 3). Both fragments were present in NCAG11 and absent in Saluda. The F2 segregation patterns for both markers followed the expected 3:1 ratio of a single dominant gene (Table 3). The inheritance of powdery mildew resistance in the F2:3 lines derived from the F2 plants involved in SSR and AFLP analyses followed the expected 1:2:1 ratio (data not shown). The two primer combinations used to generate the AFLPs of interest in this study were not among those used to construct published AFLP maps of wheat (Hazen et al., 2002; Huang et al., 2000). Thus the chromosome location of the resistance gene in NCAG11 was not identified by AFLP analysis.
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Twenty-three F2:4 lines derived from 11 homozygous resistant and 12 homozygous susceptible F2:3 lines in the Saluda x NCAG11 population were evaluated for resistance to powdery mildew in the field in 2003. There was complete agreement in the categorization of F2:4 progenies and the parental F2:3 lines with respect to powdery mildew reaction. One individual per F2:4 line and the parents were evaluated for the presence of the AFLP bands XE36M55-126 and XE38M54-196 and SSR alleles at the Xgwm332 and Xwmc525 loci. There was complete agreement between expected and observed AFLP and SSR allele presence in the resistant and susceptible progeny reflecting the tight linkage among the loci.
The resistance genes in NCA4 and NCAG11 are both located on chromosome 7AL. It is not surprising that both resistance genes were introgressed into the A genome given the homology between the A genomes of the donor species T. monococcum subsp. monococcum and T. timopheevii subsp. armeniacum with the A genome of common hexaploid wheat (Feldman, 2001). The disease scores of the two germplasms in greenhouse evaluations indicated that they contain either different genes or different alleles at a single locus (Table 1). This difference was supported by four years of evaluation in 5.1-m2 plots at Kinston where significantly different disease severity means for Saluda (6.3), NCA4 (3.3), and NCAG11 (0.25) (LSD0.05 = 0.56) were recorded. Two years of field evaluations in single 1.2-m rows indicated that the resistance genes in NCA4 and NCAG11 are different from Pm1a (Table 4). The Pm17 isoline exhibited a disease score similar to NCA4 and NCAG11, but this resistance gene was introgressed from rye (Secale cereale L.) and is located on the 1AL.1RS wheat–rye translocation segment (Heun et al., 1990). Murphy et al. (2002) reported that the resistance gene in NCAG11 was different from both Pm1a and Pm9. The Pm1 and Pm9 loci are located on chromosome 7AL separated by a map distance of 8.5 cM (Schneider et al., 1991; McIntosh et al., 1998). Five alleles (1a–1e) have been identified at the Pm1 locus (Hsam et al., 1998; Singrun et al., 2003). The single allele conferring powdery mildew resistance at Pm9 has a recessive mode of action. Singrun et al. (2004) identified an additional recessive allele tightly linked and distal to the Pm1 locus. Given the chromosomal location of the resistance genes in NCA4 and NCAG11 they could be alleles of the Pm1 or Pm9 loci, or alleles of a new locus, or loci, in a cluster of closely linked resistance genes (Chantret et al., 2000). A mean map distance of 24.6 cM between the Xgwm332 and Pm1 loci has been reported (Singrun et al., 2003, 2004; Neu et al., 2002) and Shi et al. (2003) reported a distance of 24.4 cM between Pm1 and Xbarc292. These results suggested that the resistance genes in NCA4 and NCAG11 are not alleles at the Pm1 locus. Nevertheless, flanking diploid and tetraploid germplasm introgressed with the resistance genes may suppress recombination between Pm1 and Xgwm332 in NCAG11 and Pm1 and Xbarc 292 in NCAG4. Tests for allelism and further identification of linked markers are necessary to resolve these different scenarios.
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Received for publication September 10, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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