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a Dep. of Horticulture, Univ. of Nebraska, Lincoln, NE 68583
b Dep. of Plant Pathology, Univ. of Nebraska, Lincoln, NE 68583
c Life Sciences Informatics, Monsanto Company, St. Louis, MO 63167
* Corresponding author (jsteadman1{at}unl.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: ALP, abaxial leaf pubescence • APR, adult plant resistance • BCMV, Beancommon mosaic virus • BSA, bulked segregant analysis • CBB, common bacterial blight • cM, centimorgan • FW, Fusarium wilt • GGW, golden gate wax • GN, great northern • HB, halo blight • LG, linkage group • MA, Middle American • QTL, quantitative trait locus (loci) • RAPD, random amplified polymorphic DNA • RIL, recombinant inbred line • SR, specific resistance
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Sources of resistance to bean rust have been identified (Stavely, 1988; Mmbaga and Stavely, 1988; Stavely and Pastor-Corrales, 1989; Echavez et al., 1982; Arnaud-Santana et al., 1993). Resistant bean cultivars–breeding lines have been developed and released (Stavely and Grafton, 1989; Stavely and Steinke, 1990; Stavely and McMillan, 1992; Stavely et al., 1989; Coyne et al., 1994; Stavely et al., 1994). Each breeding line possessed at least two different rust resistance genes (Mmbaga et al., 1996b). Kelly et al. (1996) described and discussed the original and revised assigned symbols for rust resistance genes in beans. Miklas et al. (2002) reported integration of identified rust resistance genes in the bean linkage map. Different patterns of specific resistance to individual races or pathotypes of U. appendiculatus have been reported as follows: single dominant genes (Coyne and Schuster, 1975; Bravo and Galvez, 1976; Kolmer and Groth, 1984; Augustin et al., 1972; Finke et al., 1986; Park et al., 1999, 2000), a single recessive gene (Zaiter et al., 1989), two genes with epistatic interaction (Finke et al., 1986), two complementary dominant genes (Grafton et al., 1985), and two independent genes (Grafton et al., 1985). The Ur-7 gene has been shown to have some valuable resistance against races 41, 44, 47, 49, 67 and 108 [Pastor-Corrales, United States Department of Agriculture (USDA)-Beltsville, personal communication]. Race 108 produces a susceptible reaction on Ur-11, which has resistance to 89 of 90 races at USDA-Beltsville.
Bulked segregant analysis (BSA) (Michelmore et al., 1991) is an efficient method to rapidly identify molecular markers linked to a specific gene using bulked DNA from F2 plants. This technique was used to detect different genes (Ur-3, Ur-5, and Ur-11) of MA origin and (Ur-4, Ur-6, and Ur-9) of Andean origin for rust resistance in beans by means of RAPD markers (Haley et al., 1993, 1994; Miklas et al., 1993; Johnson et al., 1995; Park et al., 1999, 2000). Using BSA, Jung et al. (1998) identified RAPD markers linked to the Ur-12 gene for adult plant resistance (APR) to rust and the Pu-a gene for abaxial leaf pubescence (ALP). They reported no linkage between the Ur-12 locus for APR and the Pu-a locus for ALP, thought to be associated with APR (Mmbaga and Steadman, 1992). Pyramiding monogenic resistance genes into a bean cultivar is a strategy recommended to obtain durable rust resistance (Coyne and Schuster, 1975; Mmbaga et al., 1996b). Molecular markers linked to rust resistance genes are useful to pyramid these genes into a single cultivar (Kelly, 1995). However, markers linked to the Ur-7 gene of MA origin for specific resistance (SR) to rust present in Great Northern (GN) 1140 have not been reported.
The objectives of this study were to identify RAPD markers linked to the Ur-7 gene for SR to rust race 59 by means of BSA in an F2 population from the MA common bean cross GN1140 (resistant) x GN Nebr. #1 (susceptible), position the Ur-7 gene on a genetic linkage map constructed from the MA cross GN BelNeb-RR-1 x A 55, and determine the presence or absence of these identified markers in bean cultivars–lines. The RIL population from the cross BelNeb-RR-1 x A 55 was previously used to construct a genetic linkage map with RAPD markers.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Eighty-nine F2 plants and 78 F3 families (12–16 plants per F3) with the parents GN1140 and Nebr. #1, 33 MA or Andean bean cultivars–lines, and 78 RILs with their parents BelNeb-RR-1 and A 55 were planted in the greenhouse. Two or four plants were grown per clay pot containing equal parts by volume of sand, sphagnum peat moss, vermiculite, and sharpsburg silty clay loam soil. Peters 20N:10P:20K Peat-Lite Special (Scotts-Sierra Horticultural Products Co., Marysville, OH) water soluble fertilizer was applied weekly. The chemical Orthene 75 s soluble powder (active ingredient acephate: O,S-dimethyl acetyl-phosphophoromidothioate) (Valent Co., Walnut Creek, CA) was applied biweekly to control white flies (Bemisia spp.). Approximate day/night greenhouse temperatures were 26 ± 2/22 ± 2°C in each experiment. Lengths of natural days/nights ranged from 13/11 to 15/9 h in each experiment.
Inoculation
Rust race 59, collected from the USA, was used for inoculation of 33 bean cultivars–lines as well as 89 F2 plants and 78 F3 families. Race 59 was chosen because in our preliminary screening of SR to races GN1140 was resistant to race 59, whereas Nebr. #1 was susceptible. A water suspension of approximately 2 x 104 urediniospores/mL with 40 µL/L of Tween-20 was prepared for inoculation. The abaxial leaf surfaces of two unifoliolate leaves in each plant were inoculated at 7 d after planting with the rust spore suspension by the inoculation technique of Stavely (1983). Inoculated plants were incubated in a misting chamber at 20 to 22°C for 16 h and then transferred from the chamber to greenhouse benches. Rust reactions on the unifoliolate leaves of each plant were recorded at 14 d after inoculation on the basis of the disease rating scale described by Stavely et al. (1983). Resistant reactions included no visible symptom (immune), necrotic spots without sporulation (a hypersensitive reaction), and/or small uredinia less than 300 µm in diameter. A susceptible reaction produced uredinia larger than 300 µm in diameter.
Bulked Segregant Analysis Using RAPD
Noninoculated fully expanded trifoliolate leaves of 89 F2 plants and 78 RILs as well as 33 cultivars–lines were collected at 21 d after planting. Total genomic DNA was extracted from the lyophilized leaf tissue by the method of Skroch and Nienhuis (1995). A total of 280 10-mer primers (Operon Technologies, Alameda, CA) were used for the RAPD analysis (Williams et al., 1990). Polymerase chain reactions (PCR) were performed in thin-walled glass capillary tubes in an air thermalcycler (model 1605; Idaho Technology, Idaho Falls, ID) and on 48-well plates in a Perkin-Elmer thermalcycler (model 480; Perkin-Elmer Co., Norwalk, CT). PCR protocols and the composition of the final volume of reactants were similar to those described by Skroch and Nienhuis (1995).
Two different bulked DNAs were prepared from equal volumes of standardized DNA (10 ng/µL) from eight homozygous resistant and eight homozygous susceptible F2 plants selected on the basis of F3 reaction to rust race 59, respectively. The 280 primers were used to simultaneously screen between the two different bulked DNAs from resistant and susceptible F2 plants, and between the parents GN1140 and Nebr.#1. Molecular size markers from a 100-base pair (bp) ladder (Life Technologies, Grand Island, NY) were used to estimate the length of RAPD markers. The name of each RAPD marker is derived from an ‘O’ prefix for Operon primers, the letters identifying the Operon kit, Operon primer number, and the approximate length of the marker.
Linkage Analysis
To test the genetic hypothesis of a single dominant gene controlling rust resistance, the chi-square test was used to test goodness-of-fit to a 3:1 resistant to susceptible ratio in the F2 generation or a 1:2:1 segregation of nonsegregating for rust resistance, segregating, and nonsegregating for rust susceptibility in the F3 generation. To detect segregation distortion of markers, F2 and RIL population marker data were tested for goodness-of-fit to a 3:1 or 1:1 ratio.
Because of the dominant character of RAPD markers, the linkage analysis of six coupling- or three repulsion-phase RAPD markers with the Ur-7 locus for SR to rust was separately performed on the data for 89 F2 plants of the cross GN1140 x Nebr. #1 with MAPMAKER version 3.0 (Lander et al., 1987). The linkage analysis of 90 markers containing 87 RAPD markers, one sequence characterized amplified region, the I gene for BCMV resistance and a HB resistance gene previously mapped by Ariyarathne et al. (1999) with nine new RAPD markers identified in our study was also executed on the data for the RIL population of the cross BelNeb-RR-1 x A 55 with MAPMAKER version 3.0. On the basis of a logarithm of odds (LOD) score of 3.0 and a linkage threshold of 0.4, linkage groups (LGs) were displayed by the Group command. For building LGs, a subset of markers was initially selected on the basis of LOD scores and pairwise linkages. The best linkage order within the subset was calculated by the Compare command and then, additional markers were inserted by the Try command. LOD scores of at least 2.0 were considered different between the most and second most likely position for the marker. The Ripple command was finally used to check the marker order. Map distances (cM) between ordered loci of marker and gene were calculated by recombination fractions and the Kosambi mapping function (Kosambi, 1944).
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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2 = 0.00 and P = 0.95) of number of resistant to susceptible F2 plants to race 59 was observed. It was hypothesized that a single dominant gene controlled SR to race 59. The genetic hypothesis of a single dominant gene for resistance to race 59 was confirmed in the F3 generation on the basis of a satisfactory fit to a 1:2:1 ratio (2 = 1.87 and P = 0.42) of number of families nonsegregating for resistance, segregating for resistance and susceptibility, and nonsegregating for susceptibility. A single dominant gene for SR to race 59 found here agrees with the findings of Augustin et al. (1972), who reported that the reaction to Brazilian rust race B11 was controlled by a major gene in four different F2 populations from crosses of GN1140 with susceptible bean cultivars–lines such as Gallatin 50, PI 165078, Tendergreen, and Dark Red Kidney. The symbol RB11 was assigned by Augustin et al. (1972) for the dominant gene for resistance to Brazilian rust race B11. The RB11 gene was subsequently defined as Ur-7 and recognized to be different from other rust genes by Kelly et al. (1996). Augustin et al. (1972) also found no genetic linkage between genes for plant habit and rust resistance.
RAPD Markers Linked to the Ur-7 Gene for Specific Rust Resistance
A total of 280 primers were used for the RAPD analysis of two different bulks developed from resistant and susceptible F2 plants along with their parents GN1140 and Nebr. #1. Twenty-six RAPD markers were polymorphic for the two different bulked DNAs. Sixteen displayed an amplified DNA fragment in the resistant bulk that was absent in the susceptible bulk (Fig. 1). Ten showed an amplified DNA fragment in the susceptible bulk that was absent in the resistant bulk (Fig. 2). These 26 marker fragments segregated in the F2 population of the cross GN1140 x Nebr. #1. Of these 26 markers, nine were linked to the Ur-7 gene. A goodness-of-fit to a 3:1 ratio for band presence to band absence for each of the nine markers was observed in 89 F2 plants (Table 1).
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These nine markers were also polymorphic between the parents BelNeb-RR-1 and A 55 that were utilized to create the RIL population from which the RAPD marker-based genetic linkage map was constructed. Six coupling-phase markers displayed an amplified DNA fragment in the BelNeb-RR-1 parent possessing at least three different rust resistance genes Ur-5, Ur-6, and Ur-7. Three repulsion-phase markers displayed an amplified DNA fragment in the A 55 parent susceptible to rust. Examples of coupling-phase marker OAD12.550 and repulsion-phase marker OAB18.650 are shown in Fig. 1 and 2. All nine markers also segregated in the RIL population of the cross BelNeb-RR-1 x A 55. A goodness-of-fit to a 1:1 ratio for band presence to band absence for each of the nine markers was observed in the RIL population (Table 1). On the basis of linkage analysis of 90 formerly mapped markers with nine new RAPD markers identified here, all nine markers linked to the Ur-7 gene were classified into one LG and located within a distance of 8.1 cM on LG 11 of the existing linkage map (Fig. 3). Important rust resistance genes such as Ur-3, Ur-6, and Ur-11 are located on LG 11 of the core bean map, whereas other rust genes Ur-9, Ur-5, and Ur-4 are located on LGs 1, 4, and 6, respectively (Miklas et al., 2002). However, the Ur-7 gene is located in a different region on LG 11 with respect to the other important rust resistance genes, on the basis of the result of Miklas et al. (2002). Also, the Ur-BAC 6 gene is located on LG 11, but the distance between the Ur-BAC 6 and the Ur-7 genes is more than 50 cM (Miklas et al., 2002).
The presence or absence of two coupling-phase markers OAD12.550 and OAF17.900 linked to the Ur-7 gene for SR to rust in the F2 population was investigated in 21 MA and 12 Andean common bean cultivars–breeding lines (Table 2). The presence of these markers was associated with all MA cultivars–lines resistant to rust race 59 except BelDakMi-RR-1 and Chase. All MA susceptible cultivars–lines lacked the marker fragments. Two resistant lines BelDakMi-RR-1 and Chase possessed a different rust resistance gene. All Andean cultivars–lines, regardless of response to race 59, lacked the marker fragments. This would be expected because the Ur-7 gene is from the MA gene pool, and these markers are closely linked with the gene. Thus, these coupling-phase markers OAD12.550 and OAF17.900 could be useful in breeding and selecting for rust resistance in the MA bean germplasm.
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Kelly et al. (1996) indicated that two independently identified rust resistance genes, the Ur-6 gene present in the Olathe cultivar and the Ur-7 gene present in the GN1140 cultivar, might be the same. Grafton et al. (1985) originally assigned the symbol Ura for a single gene for resistance that is known to be independent of other rust resistance genes. Stavely et al. (1983) reported that the Golden Gate Wax (GGW) cultivar possessed the Ura gene for rust resistance originally identified by Ballantyne (1978). Kelly et al. (1996) subsequently reassigned the Ura gene as Ur-6 present in the Olathe and GGW cultivars. Both Olathe and GGW were resistant to race 59 in our tests (Table 2). Thus, we needed to determine if coupling-phase markers linked to the Ur-7 gene identified here were also present in the two cultivars containing the Ur-6 gene. All the coupling-phase markers were present in the Olathe cultivar as well as pintos BelDak-RR-1 and Bill Z, another source of Ur-6 (M. Brick, Colorado State Univ., personal communication) (Table 2). They were absent in the GGW cultivar and another source of Ur-6, BelDakMi-RR-1. These coupling-phase markers also segregated in an F2 population of the bean cross Olathe x GN Nebr.#1 sel.27. On the basis of linkage analysis of the coupling-phase markers with the Ur-6 gene, the markers were not linked to the Ur-6 gene in the population (unpublished data). These results support the hypothesis that the Ur-6 and Ur-7 genes are not the same.
Pyramiding monogenic resistance genes has been proposed as an effective and economic strategy to develop stable and durable resistance to rust. Kelly (1995) reported that pyramiding three major genes (Ur-3, Ur-4, and Ur-5) resulted in resistance to 63 of the 65 bean rust races reported in the USA. However, because of epistasis between rust resistance genes (Kolmer and Groth, 1984), the pyramiding of monogenic resistance genes can be a time-consuming and expensive procedure. Molecular markers linked to major genes for rust resistance could be useful when several resistance genes from different sources are transferred into a susceptible bean cultivar (Kelly, 1995). For the MA gene pool, coupling-phase RAPD markers linked to three different genes for rust resistance have been developed: OI19.460 linked to Ur-5 in Mexico 309 (Haley et al., 1993), OK14.620 linked to Ur-3 in Aurora (Haley et al., 1994), and OAC20.490 linked to Ur-11 in PI181996 (Johnson et al., 1995). Similarly, three different genes from the Andean gene pool for rust resistance have tightly linked coupling-phase RAPD markers: OA14.1100 linked to Ur-4 in Early Gallatin (Miklas et al., 1993), OAG15.300 linked to Ur-6 in Olathe (Park et al., 2000), and OA4.1050 linked to Ur-9 in PC-50 (Park et al., 1999). Jung et al. (1998) mapped O13.1350 linked to the Ur-12 gene for APR and G3.1150 linked to the Pu-a gene for ALP. The ALP trait was thought to be associated with race-nonspecific APR (Mmbaga and Steadman, 1992). Stable rust resistance may be improved by incorporating the APR and ALP traits. The coupling-phase RAPD markers linked to the Ur-7 gene of MA gene pool identified here, along with the markers linked to the above genes from other germplasm, could be utilized to develop a P. vulgaris cultivar or line with different genes pyramided for enhanced broad resistance to rust. Park et al. (1999) also suggested that recombining resistance genes from both Andean and MA gene pools should provide more durable resistance to rust.
Genetic Relationships of the Ur-7 Gene with Other Disease Traits
Using the RILs from the cross BelNeb-RR-1 x A 55, three quantitative trait loci (QTL) for leaf resistance to CBB on LGs 1, 9, and 10 and two QTL affecting pod resistance to CBB on LGs 2 and 10 were identified by Ariyarathne et al. (1999). They reported six QTL controlling HB resistance to two different bacterial strains on LGs 2, 3, 4, 5, 9, and 10 for the first time in beans. They also mapped the I gene for resistance to BCMV on LG 2. A major QTL on LG 10 that explained more than 60% of the phenotypic variation for resistance to FW was found by Fall et al. (2001). However, all molecular markers linked to the Ur-7 gene for SR to rust were located on LG 11 of the map (Fig. 3). Thus, there was no genetic relationship between the newly identified gene and the previously mapped resistance genes–QTL.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication July 22, 2002.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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  S. O. Park, J. R. Steadman, D. P. Coyne, and K. M. Crosby Development of a Coupling-Phase SCAR Marker Linked to the Ur-7 Rust Resistance Gene and Its Occurrence in Diverse Common Bean Lines Crop Sci., January 16, 2008; 48(1): 357 - 363. [Abstract] [Full Text] [PDF]
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S. O. Park, D. P. Coyne, J. R. Steadman, K. M. Crosby, and M. A. Brick RAPD and SCAR Markers Linked to the Ur-6 Andean Gene Controlling Specific Rust Resistance in Common Bean Crop Sci., September 1, 2004; 44(5): 1799 - 1807. [Abstract] [Full Text] [PDF]
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