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RAPD and SCAR markers linked to the Ur-6 Andean gene controlling specific rust resistance in common bean.

BEAN RUST is an important disease resulting in reduced bean yield and quality in many parts of the world (Stavely and Pastor-Corrales, 1989). It consistently causes yield reductions ranging from 18 to 100% in dry and snap beans (Stavely and Pastor-Corrales, 1989; Lindgren et al., 1995). The pathogenic variability of the fungus is broad, with over 300 races or pathotypes recognized (Stavely and Pastor-Corrales, 1989; Mmbaga et al., 1996a). Cultural practices such as crop rotation, intercropping, elimination of plant debris, adjustment of planting dates, and blending heterogeneous landrace cultivars can reduce the disease severity (Plaut and Berger, 1981; Mundt and Brophy, 1988; Stavely and Pastor-Corrales, 1989; Boudreau and Mundt, 1992). However, the use of host plant resistance is the most economic and environmentally sustainable method for controlling bean rust (Coyne and Schuster, 1975; Mmbaga et al., 1996b).

Sources of resistance to bean rust have been identified (Mmbaga and Stavely, 1988; Stavely and Pastor-Corrales, 1989; Echavez et al., 1982; Arnaud-Santana et al., 1993). Resistant bean cultivars and breeding lines have been developed and released (Wood and Keenan, 1982; Stavely and Steinke, 1990; Stavely and McMillan, 1992; 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. Different patterns of specific resistance to individual rust races or pathotypes 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, 2003), 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).

Bulked segregant analysis (BSA) (Michelmore et al., 1991) is an efficient method to rapidly identify molecular markers linked to a specific gene using DNA bulks from [F.sub.2] plants. This technique was used to detect four different genes (Ur-3, Ur-5, Ur-7, and Ur-11) of Middle American (MA) origin and two genes (Ur-4 and Ur-9) of Andean origin for rust resistance in beans using random amplified polymorphic DNA (RAPD) markers (Haley et al., 1993, 1994; Miklas et al., 1993; Johnson et al., 1995; Park et al., 1999, 2003). Miklas et al. (2002) reported integration of identified rust resistance genes in the bean linkage map. Three important rust resistance genes such as Ur-3, Ur-7, and Ur-11 of MA origin are located on linkage group (LG) 11 (Miklas et al., 2002; Park et al., 2003), while other rust genes Ur-9, Ur-5 and Ur-4 are located on LGs 1, 4, and 6, respectively (Miklas et al., 2002). Pyramiding monogenic resistance genes into a bean cultivar is a strategy recommended to obtain durable rust resistance (Mmbaga et al., 1996b). Those markers linked to rust resistance genes have been utilized to pyramid these genes into a single cultivar, as suggested by Kelly (1995).

Grafton et al. (1985) reported a single dominant resistance gene of Andean origin present in Olathe. This rust resistance gene, along with other sources, was utilized to develop resistant pinto and great northern (GN) bean breeding lines (Stavely and Grafton, 1989; Stavely et al., 1989, 1992). McClean et al. (1994) initially identified RAPD marker OV12.950 linked to Ur-6 for resistance to race 47 at 10.4 cM in a pinto bean cross. On the basis of this result Miklas et al. (2002) mapped Ur-6 on LG 11 of the core bean map. However, markers tightly linked to Ur-6 of Andean origin for specific resistance (SR) to rust present in Olathe have not been reported.

The objective of this study was to identify RAPD markers tightly linked to Ur-6 for SR to race 51 by BSA in an [F.sub.2] population from the MA common bean cross Olathe (resistant) x GN Nebr.#1 sel.27 (susceptible). Merits of sequence characterized amplified region (SCAR) markers over RAPDs have been reported (Paran and Michelmore, 1993; Melotto et al., 1996). Thus, our additional goal was to convert the most tightly linked RAPD marker to Ur-6 into a SCAR marker on the basis of a specific forward and reverse 24-mer primer pair. We then determined the presence or absence of these identified RAPD and SCAR markers in 70 MA and Andean bean genotypes with or without Ur-6.

MATERIALS AND METHODS

Plant Materials

One hundred [F.sub.2] plants derived from the common bean cross Olathe x GN Nebr.#1 sel.27 were developed to score Ur-6 segregation in a greenhouse, Department of Horticulture, University of Nebraska-Lincoln in winter 1997. The pinto seeded Olathe of the MA background possesses Ur-6 ([Ur.sub.a]) of Andean origin for SR to rust (Grafton et al., 1985; Kelly et al., 1996), while the white seeded Nebr.#1 sel.27 does not have Ur-6. Seventy-eight recombinant inbred lines (RILs) derived from the MA common bean cross BelNeb-RR-1 x A 55 were previously developed by single-seed-descent (Ariyarathne et al., 1999). Stavely et al. (1989) developed GN BelNeb-RR-1 with at least three different rust resistance genes Ur-5 (B-190), Ur-6 ([Ur.sub.a]), and Ur-7 ([R.sub.B11]). We used this RIL population for the mapping of Ur-6.

One hundred [F.sub.2] plants and 88 [F.sub.3] families (12 to 16 plants per [F.sub.3]), along with their parents Olathe and Nebr.#1 sel.27, were planted ill the greenhouse on 16 Feb. 1998 and 18 Aug. 1998, respectively. Twelve of the 100 [F.sub.3] families were not planted because of a lack of seeds. Fifty-seven MA and 13 Andean common bean cultivars-breeding lines were also planted in a completely randomized design with two replications in the greenhouse on 4 Feb. 2003. 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 tabaci (Gennadius)]. Approximate day/night greenhouse temperatures were 26 [+ or -] 2/22 [+ or -] 2[degrees]C in each experiment. Lengths of natural days/nights ranged from 13/11 to 15/9 h in each experiment.

Inoculation

Race 51 of the bean rust pathogen, collected from the USA, was used for inoculation of 57 MA and 13 Andean bean cultivars-lines as well as 100 [F.sub.2] plants and 88 [F.sub.3] families. Race 51 was chosen because in our preliminary screening of SR to several races of the rust pathogen, Olathe was resistant to race 51, whereas Nebr.#1 sel.27 was susceptible. A water suspension of approximately 2 x [10.sup.4] urediniospores/mL with 40 [micro]L/L of Tween-20 [polyoxyethylene (20) sorbitan monolaurate] 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 using the inoculation technique of Stavely (1983). Inoculated plants were incubated in a misting chamber at 20 to 22[degrees]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 using 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 [micro]m in diameter. A susceptible reaction produced uredinia larger than 300 [micro]m in diameter.

Bulked Segregant Analysis Using RAPD

Noninoculated fully expanded trifoliolate leaves of 100 [F.sub.2] plants along with their parents as well as 70 bean cultivars and breeding lines were collected at 21 d after planting. Total genomic DNA was extracted from the lyophilized leaf tissue using the method of Skroch and Nienhuis (1995). A total of 680 random 10-mer primers (Operon Technologies, Alameda, CA) were used for the RAPD analysis (Williams et al., 1990). Polymerase chain reactions (PCR) were performed on 96-well plates in a MJ Research thermocycler (model PTC-0100; MJ Research, Waltham, MA). PCR protocols and the composition of the final volume of reactants were similar to those described by Skroch and Nienhuis (1995). Molecular size markers from a 100-base pair 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 Opcron kit, Operon primer number, and the approximate length of the marker.

Two different DNA bulks were prepared from equal volumes of standardized DNA (10 ng/[micro]L) from eight homozygous resistant and eight homozygous susceptible [F.sub.2] plants selected on the basis of [F.sub.3] phenotypic data for the reaction to race 51 of the bean rust pathogen, respectively. The 680 primers were used to simultaneously screen between the two different DNA bulks from resistant and susceptible [F.sub.2] plants, and between the parents Olathe and GN Nebr.#1 sel.27. Twenty-six primers generated marker polymorphisms between the DNA bulks from resistant and susceptible plants, and they were tested subsequently in the [F.sub.2] population of the cross Olathe and Nebr.#1 sel.27. Two primers were tested in 70 bean cultivars/breeding lines for determining the presence or absence of coupling-phase markers linked to Ur-6.

Converting RAPD to SCAR Markers

To develop a SCAR marker for the RAPD marker OBC06.300, the DNA fragment of the RAPD marker was excised and purified with the GENECLEAN II Kit (Q-BIO gene, Carlsbad, CA). Insertion of the purified RAPD fragment into the pCR 2.1- TOPO and cloning of the transformed plasmid were conducted with the TOPO TA Cloning Kit (Invitrogen, Carlsbad, California). The cloned plasmid was har vested using the GenElute Plasmid Miniprep Kit (Sigma, St. Louis, MO). The RAPD fragment was sequenced with the M13 reverse and forward primers at the DNA sequencing and synthesis facility of the Iowa State University Office of Biotechnology (Ames, IA). A specific forward and reverse 24-mer primer pair was designed on the basis of the forward and reverse sequences of the RAPD fragment. The forward and reverse primer pair was synthesized at the Operon Technologies. PCR was performed on 96-well plates in the MJ Research thermocycler. PCR protocols and the composition of the final volume of reactants were similar to those described by Rubio et al. (2001) with an annealing temperature of 65[degrees]C. The specific forward and reverse 24-mcr primer pair was tested in the [F.sub.2] population of the cross Olathe and Nebr.#1 sel.27. The primer pair was also tested in 70 MA and Andean bean cultivars/lines for determining the presence or absence of the SCAR marker linked to Ur-6.

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 [F.sub.2] generation or a 1:2:1 segregation of nonsegregating for rust resistance, segregating, and nonsegregating for rust susceptibility in the [F.sub.3] generation. To detect segregation distortion of markers, the [F.sub.2] population marker data was tested for goodness-of-fit to a 3:1 ratio.

Because of the dominant character of RAPD and SCAR markers, the linkage analysis of seven coupling- or five repulsion-phase markers with the Ur-6 locus for SR to rust was separately performed on the data for 100 [F.sub.2] plants of the cross Olathe x Nebr.#1 sel.27 by MAPMAKER version 3.0 (Lander et al., 1987). On the basis of a logarithm of odds (LOD) score of 3.0 and a linkage threshold of 0.4, LGs were displayed by the Group command. To establish a LG, 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 (centimorgan, cM) between ordered loci of marker and gene were calculated using recombination fractions and the Kosambi mapping function (Kosambi, 1944).

RESULTS AND DISCUSSION

Inheritance of the Reaction to Race 51

Clear separation for the disease rating of the parents Olathe and GN Nebr.#1 sel.27 to race 51 of the bean rust pathogen was observed in the greenhouse test. Large uredinia more than 800 [micro]m in diameter were found on the unifoliolate leaves of all plants of Nebr.#1 sel.27, indicating that Nebr.#1 sel.27 was susceptible to race 51. In contrast, Olathe was resistant to race 51 and showed no disease symptoms on the inoculated unifoliolate leaves of all plants. [F.sub.1] plants derived from the cross Olathe x GN Nebr.#1 sel.27 were resistant to race 51. A goodness-of-fit to a 3:1 ratio ([chi square] = 0.01 and P = 0.91) of number of resistant to susceptible [F.sub.2] plants to race 51 was observed, it was hypothesized that a single dominant gene controlled SR to race 51. The genetic hypothesis of a single dominant gene for resistance to race 51 was confirmed in the [F.sub.3] generation based on a satisfactory fit to a 1:2:1 ratio ([chi square] = 1.35 and P 0.51) of number of families nonsegregating for resistance, segregating for resistance and susceptibility, and nonsegregating for susceptibility.

A single dominant gene for SR to race 51 found here agrees with the findings of Grafton et al. (1985) and McClean et al. (1994), who reported that the reaction to races 44 and 47 of the rust pathogen was controlled by a single dominant gene in [F.sub.2] populations from two pinto bean crosses Olathe x UI-114 and Sierra x Olathe. However, Grafton et al. (1985) also reported two complementary dominant genes controlling resistance to race 44 in an [F.sub.2] population from a cross Olathe x T-39 and two independent genes conferring resistance to races 44 and 52 in an [F.sub.2] population from a cross Olathe x Aurora. The symbol [Ur.sub.a] was assigned by Grafton et al. (1985) for the dominant resistance gene of Andean origin present in Olathe of the MA background. The [Ur.sub.a] gcnc was subsequently defined as Ur-6 by Kelly et al. (1996). It is known to be independent of other rust resistance genes and is resistant to 58 of 93 races available at USDA-Beltsville (Pastor-Corrales, USDA-Beltsville, personal communication).

RAPD Markers Linked to Ur-6 for Specific Rust Resistance

A total of 680 primers were used for the RAPD analysis of two different bulks developed from resistant and susceptible [F.sub.2] plants along with their parents Olathe and Nebr.#1 sci.27. Thirty-two RAPD markers were polymorphic for the two different DNA bulks. Twenty displayed an amplified DNA fragment in the resistant bulk that was absent in the susceptible bulk (Fig. 1a). Twelve showed an amplified DNA fragment in the susceptible bulk that was absent in the resistant bulk (Fig. 1c). These 32 marker fragments segregated in the [F.sub.2] population of the cross Olathe x Nebr.#1 sel.27. Of the 32 markers, 11 were identified to be linked to Ur-6 on the basis of linkage analysis. A goodness-of-fit to a 3:1 ratio for band presence to band absence for each of the 11 markers was observed in 100 [F.sub.2] plants (Table 1).

[FIGURE 1 OMITTED]

The integrated location of the Ur-6 locus for SR to rust and the loci of six coupling-phase RAPD markers that displayed an amplified DNA fragment in the resistant bulk is shown in Fig. 2. This LG included seven loci spanning a length of 21 cM. Two markers OBC06.300 and OAG15.300 were detected as flanking markers tightly linked to Ur-6 at distances of 1.3 and 2.0 cM, respectively. These flanking markers would be effective in selecting Ur-6. Three coupling-phase markers OAU03.500, OAB14.450, and OAT15.650 were also linked to the Andean gene at distances of 3.5 to 6.3 cM. The combined location of the Ur-6 locus and the loci of five repulsion-phase RAPD markers that displayed an amplified DNA fragment in the susceptible bulk is also presented in Fig. 2. The LG included six loci spanning a length of 43 cM. Repulsion-phase marker OAY15.200 was the most closely linked to Ur-6 controlling SR to rust among the five markers at a distance of 7.7 cM. Two markers OAL17.600 and OBF18.450 were also linked to the Andean origin gene at distances of 9.8 and 12.4 cM on the LG, respectively.

[FIGURE 2 OMITTED]

The tight linkages of these coupling- and repulsion-phase RAPD markers with Ur-6 of Andean origin controlling SR to rust in Olathe of the MA background identified here could be more useful in selecting the gene than the finding of McClean et al. (1994), who reported that one RAPD marker OV12.950 was linked to Ur-6 for resistance to race 47 at a distance of 10.4 cM on a partial RAPD marker-based linkage map containing 140 markers constructed using a RIL population from the pinto bean cross Sierra having the Ur-3 MA gene x Olathe.

Of the five repulsion-phase RAPD markers linked to Ur-6 identified in the F2 population from the cross of Olathe x Nebr.#1 sel.27, two markers OAB18.650 and G5.500 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 linkage map was constructed (Ariyarathne et al., 1999). These two repulsion-phase markers, very loosely linked to the Andean gene in the [F.sub.2] population, displayed an amplified DNA fragment in the A 55 parent susceptible to rust, whereas they were absent in the BelNeb-RR-1 parent resistant to rust. The two markers OAB18.650 and G5.500 were observed to be located within a distance of 4 cM on LG 11 of the RAPD marker-based linkage map constructed from the RIL population from the cross GN BelNeb-RR-1 x A 55 (Fig. 2), suggesting that Ur-6 was positioned on LG 11 of the map. However, six coupling-phase RAPD markers linked to Ur-6 found in the [F.sub.2] population here were not noted on the RAPD linkage map.

The Ur-6 location found here confirms the finding of Miklas et al. (2002), who reported that the Andean gene was mapped on LG 11 of the core bean linkage map, on the basis of the result of McClean et al. (1994). Important rust resistance genes of MA origin such as Ur-3, Ur-7, and Ur-11 are located on LG 11 of the core bean map, while other rust genes Ur-9 of Andean origin, Ur-5 of MA origin, Ur-4 of Andean origin, and Ur-12 are located on LGs 1, 4, 6, and 7, respectively (Miklas et al., 2002; Park et al., 2003). However, Ur-6 is located in a different region on LG 11 with respect to the other important rust resistance genes, on the basis of the results of Miklas et al. (2002) and Park et al. (2003). Thus, a total of five rust resistance genes including four of MA origin and one of Andean origin have been mapped on LG 11 of the core bean map.

Development of a SCAR Marker Linked to Ur-6

The coupling-phase RAPD marker OBC06.300 tightly linked to Ur-6 at a distance of 1.3 cM identified in the [F.sub.2] population from the cross Olathe x Nebr.#1 sel.27 was converted into a SCAR marker on the basis of the specific forward and reverse 24-mer primer pair. The size of the RAPD fragment OBC06.300 was 308 bp based on the sequence data obtained from the DNA sequencing and synthesis facility of the Iowa State University. For generating the SCAR marker, we designed the specific forward and reverse 24-mer primer pair based on the sequence of the RAPD fragment OBC06.300. The sequence of the forward primer was 5'-GAAGGCGAGAAGAAAAAGAAAAAT-3', while that of the reverse primer was 5'-GAAGGCGAGAG CACCTAGCTGAAG-3'. The underlined sequences were the original 10-mer sequence of the OBC06 primer. Melting temperatures of the forward and reverse primers were 58 and 67[degrees]C, respectively.

The marker SOBC06.308, the name of the SCAR marker amplified with the specific forward and reverse primer pair, is shown in Fig. 1b. The marker SOBC 06.308 was present in the resistant parent Olathe and the DNA bulk from resistant F2 plants, whereas it was absent in the susceptible parent GN Nebr.#1 sel.27 and the DNA bulk from susceptible [F.sub.2] plants. The SCAR marker fragment segregated in the [F.sub.2] population of the cross Olathe x GN Nebr.#1 sel.27. A goodness-of-fit to a 3:1 ratio for band presence to band absence for the marker 8OBC06.308 was observed in 100 [F.sub.2] plants (Table 1). The integrated location of the Ur-6 locus and the loci of six coupling-phase markers including the SCAR marker is shown in Fig. 2. The marker SOBC 06.308 showed no recombination with the RAPD marker OBC06.300 in the [F.sub.2] population, and thus, the SCAR and RAPD markers were observed at the same locus on the LG. The marker SOBC06.308 was also closely linked to Ur-6 at 1.3 cM. The results confirm that the SCAR marker SOBC06.308 was derived from the RAPD marker OBC06.300. This is the first report of a coupling-phase SCAR marker linked to Ur-6 in common bean.

No recombination between the RAPD marker OBC06. 300 and the SCAR marker SOBC06.308 was expected. However, Melotto et al. (1996) observed a few recombinations between the RAPD marker OW13.690 and the SCAR marker SW13 linked to the 1 gene in a N84004/ Michelite population. Adam-Blondon et al. (1994) reported a codominant SCAR marker linked to the Are gene for resistance to anthracnose in bean, caused by Colletotrichum lindemuthianum (Sacc. & Magnus) Lams.-Scrib. For rust resistance, Correa et al. (2000) developed SCAR markers SCARBA08 and SCARF10 linked to Ur-Ouro Negro at 4.3 and 6.0 cM, respectively, on LG 4 of the core map.

Survey of RAPD and SCAR Markers in Diverse Bean Germplasm

We investigated the presence or absence of the SCAR marker SOBC06.30g as well as RAPD markers OBC06. 300 and OAG15.300 in 33 MA pinto bean cultivars breeding lines (Table 2). In combination with Ur-3 or Ur-11 of MA origin, Ur-6 was widely utilized in developing rust resistant pinto bean breeding lines including two BelDak (Stavely and Grafton, 19891 and six BelDakMi breeding lines (Stavely et al., 1992). All BelDak and BellDakMi bean lines were observed to be resistant to race 51. The presence of the two RAPD and one SCAR markers was associated with BelDak-RR-1 and 2 possessing Ur-6. Also, these three marker fragments were amplified in BelDakMi-RR-1, 2, 5, 10, 14, and 18 (genotype = Ur-6), while no marker fragment was amplified in BelDakMi-RR-4 (genotype = ur-6). Along with either Ur-5 or Ur-11, this Andean gene is currently being used to develop rust resistant pinto bean breeding lines in Colorado bean breeding programs. The presence of the markers was consistently associated with all 10 Colorado lines mostly resistant to race 51. The gene was incorporated into several elite pinto bean cultivars such as Apache, Bill Z, Burke, Topaz, and Kodiak (Wood et al., 1989). The RAPD and SCAR marker fragments were present in the five bean cultivars resistant to race 51. Other susceptible pinto cultivars or lines without the gene, except UI-111, lacked the marker fragments. These results would be expected because of the tight linkage of the markers with the gene. Thus, the RAPD and SCAR markers could be useful in breeding and selecting the Andean gene for rust resistance in MA pinto bean germplasm.

The three markers were also surveyed in 24 MA GN, black, navy, and other beans (Table 2). Rust resistant lines GN BelNeb, GN BelMiNeb, and navy BelMiDak carrying at least two different rust resistance genes were developed (Stavely et al., 1989, 1994). Particularly, Ur-6 was incorporated into BelNeb-RR-1 and BeIMiNeb-RMR-4. Five BelNeb, BelMiNeb, and BelMiDak lines were noted resistant to race 51. The marker fragments were present in GN BelMiNeb-RMR-4, while, because of recombinations of the gene with the markers, they were absent in GN BelNeb-RR-1. Other MA beans without the gene, mostly susceptible to race 51, lacked the marker fragments.

We conducted the survey of the RAPD and SCAR markers in 13 Andean beans (Table 2). Golden Gate Wax (GGW), resistant to race 51, possessed the coupling-phase markers tightly linked to Ur-6. This result is consistent with the finding of Stavely et al. (1983), who reported that the wax bean cultivar also possessed Ur-6 originally identified by Ballantyne (1978). However, the three markers were also present in six Andean cultivars without Ur-6. The marker fragments were absent in other six susceptible Andean beans lacking the gene. This would be expected because the gene is originally from the Andean gene pool. Miklas et al. (1993) and Haley et al. (1993) reported similar results that Andean beans lacking Ur-4 had a marker linked to the Andean gene, and MA beans without Ur-5 possessed a marker linked to the MA gene. Survey information of the remaining coupling-phase linked RAPD markers, except OAC03.600, to Ur-6 in 70 MA and Andean beans was similar to that of the most closely linked RAPD and SCAR markers, and thus, was not included.

The RAPD and SCAR markers were generally regarded as gene pool specific on the basis of the analysis of our marker survey (Table 2). Miklas et al. (1993) and Park et al. (2003) reported a gene pool specificity and usefulness of their identified markers for Ur-4 and Ur-7 in MA bean germplasm, respectively. A race specificity was also observed within the Andean gene pool (Haley et al., 1993). However, Haley et al. (1994) and Mclotto et al. (1996) reported no gene pool or race specificity on the basis of marker surveys in a MA and Andean bean collection, and utility of their linked markers for Ur-3 and 1 for BCMV resistance across MA and Andean bean gene pools.

Grafton et al. (1985) reported an additional rust resistance gene [Ur.sub.c] present in Olathe. Kelly et al. (1996) assumed that two rust resistance genes, [Ur.sub.c] and Ur-7, present in GN1140 were the same. Park et al. (2003) reported that two coupling-phase markers, OAD12.550 and OAF17.900, that cosegregated with Ur-7 on LG 11 were present in Olathe, whereas they were absent in GGW possessing Ur-6. The markers were not linked tn Ur-6 in the [F.sub.2] population of the cross Olathe x Nebr.#1 sel.27 (Park et al., 2003). Our RAPD and SCAR markers on LG 11 amplified from Olathe and GGW were absent in GN1140 carrying Ur-7 as well as US-5, from which the rust resistance of GN1140 was derived (Table 2). Thus, we conclude that Ur-6 and Ur-7 are distinct and loosely linked on LG 11. This marker information surveyed in key cultivars is indirect evidence that [Ur.sub.c] and Ur-7 are the same gene. Olathe, Bill Z, and BelDak-RR-1 having Ur-6 are candidates to have Ur-7 because of the presence of the markers for Ur-7, whereas GGW and BelDakMi-RR-1, which possess Ur-6, lack Ur-7.

For the MA gene pool, coupling-phase RAPD markers linked to four different rust resistance genes 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), OAC20.490 linked to Ur-11 in PI181996 (Johnson et al., 1995), and OAD12.550 linked to Ur-7 in GN1140 (Park et al., 2003). Similarly, two different rust resistance genes from the Andean gene pool have tightly linked coupling-phase RAPD markers: OA14.1100 linked to Ur-4 in Early Gallatin (Miklas et al., 1993) and OA4.1050 linked to Ur-9 in PC-50 (Park et al., 1999).

Kelly (1995) reported that pyramiding three major rust resistance genes such as Ur-3, Ur-4, and Ur-5 resulted in resistance to 63 of 65 bean races. The coupling-phase RAPD and SCAR markers linked to Ur-6 of Andean origin in Olathe and GGW identified here, along with the markers linked to the above genes, could be utilized to develop a P. vulgaris cultivar or line with different genes pyramided for enhanced broad rust resistance. Recombining resistance genes from both Andean and MA gene pools should provide more durable resistance to rust (Park et al., 1999).

Abbreviations: BCMV, Bean common mosaic virus; BSA, bulked segregant analysis; GGW, golden gate wax; GN, great northern; LG, linkage group; MA, Middle American; RAPD, random amplified polymorphic DNA; RIL, recombinant inbred line; SCAR, sequence characterized amplified region; SR, specific resistance.
Table 1. The chi-square tests for segregation of RAPD and SCAR
fragments for seven coupling-phase and five repulsion-phase markers
on linkage group 11 in an [F.sub.2] population from the Middle
American common bean cross Olathe (resistant to race 51 of the bean
rust pathogen) x Nebr.#1 set. 27 (susceptible to race 51).

                             Number of [F.sub.2]
                                   plants
RAPD and         Linkage                            Expected
SCAR markers      phase      Presence    Absence     ratio

OBC06.300       coupling        76         24         3:1
OAG15.300       coupling        76         24         3:1
OAU03.500       coupling        71         29         3:1
OAT15.650       coupling        75         25         3:1
OAB14.450       coupling        72         28         3:1
OAC03.600       coupling        75         25         3:1
SOBC06.308      coupling        76         24         3:1
OAY15.200       repulsion       59         29         3:1
OAL17.600       repulsion       58         30         3:1
OBF18.450       repulsion       59         29         3:1
OAB18.650       repulsion       58         30         3:1
G5.500          repulsion       60         28         3:1

RAPD and
SCAR markers    [chi square]        P

OBC06.300           0.01          0.91
OAG15.300           0.01          0.91
OAU03.500           0.65          0.42
OAT15.650           0.01          0.91
OAB14.450           0.33          0.56
0AC03.600           0.01          0.91
SOBC06.308          0.01          0.91
OAY15.200           2.56          0.11
OAL17.600           3.41          0.06
OBF18.450           2.56          0.11
OAB18.650           3.40          0.06
G5.500              1.84          0.18

Table 2. Presence (+) or absence (-) of two coupling-phase RAPD
markers OBC06.300 and OAG15.300, and the SCAR marker SOBC06.308
tightly linked to Ur-6 in Middle American (MA) or Andean (A) bean
cultivars/breeding lines resistant (R) or susceptible (S) to race
51 of the bean rust pathogen.

    Cultivar-breeding line
                                   Rust resistance     Reaction
Entry                    Origin         gene          to race 51

Pinto Olathe               MA       6                     R
Pinto Apache               MA       3 & 6                 R
Pinto Bill Z               MA       6                     R
Pinto Burke                MA       3 & 6                 R
Pinto Kodiak               MA       3 & 6                 R
Pinto Topaz                MA       6                     R
Pinto Chase                MA       3                     S
Pinto Fiesta               MA                             S
Pinto Othello              MA                             S
Pinto P114                 MA                             S
Pinto P650                 MA                             S
Pinto UI-111               MA                             S
Pinto US-5                 MA       7                     R
Pinto US-14                MA       7                     R
Pinto BelDak-RR-1          MA       3 & 6                 R
Pinto BelDak-RR-2          MA       3 & 6                 R
Pinto BelDakMi-RR-1        MA       6 & 11                R
Pinto BelDakMi-RR-2        MA       6 & 11                R
Pinto BelDakMi-RR-4        MA       11                    R
Pinto BelDakMi-RR-5        MA       6 & 11                R
Pinto BelDakMi-RMR-10      MA       6 & 11                R
Pinto BelDakMi-RMR-14      MA       3, 6, & 11            R
Pinto BelDakMi-RMR-18      MA       3, 4, 6, & 11         R
Pinto CO 12512             MA       6 & 11                R
Pinto CO 12515             MA       6 & 11                R
Pinto CO 12518             MA       6 & 11                R
Pinto CO 12555             MA       6 & 11                R
Pinto CO 12650             MA       5 & 6                 R
Pinto CO 12652             MA       5 & 6                 R
Pinto CO 12653             MA       5 & 6                 R
Pinto CO 12655             MA       5 & 6                 R
Pinto CO 12663             MA       5 & 6                 R
Pinto CO 12783             MA       6 & 11                S
GN GN1140                  MA       7                     R
GN Harris                  MA                             S
GN Marquis                 MA                             S
GN Valley                  MA                             S
GN Nebr.#1                 MA                             S
GN Nebr.#1 sel.27          MA                             S
GN P#44                    MA                             S
GN P#59                    MA                             S
GN P#92                    MA                             S
GN BelNeb-RR-1             MA       5, 6, & 7             R
GN BelMiNeb-1112-1         MA       4 & 11                R
GN BelMiNeb-RMR-4          MA       4, 6, & 11            R
GN BelMiNeb-RMR-7          MA       3, 4, & 11            R
Black HT 7719              MA                             S
Black A 55                 MA                             S
Navy Seafarer              MA                             S
Navy Midland               MA                             S
Navy BelMiDak-RR-8         MA       4 & 11                R
Aurora                     MA       3                     S
Ecuador 299                MA       3                     S
Mex 309                    MA       5                     S
Viva                       MA                             S
BAC 6                      MA       BAC 6                 S
Chichara                   MA                             S
Golden Gate Wax            A        6                     R
Early Gallatin             A        4                     R
Brown Beauty               A        4                     R
Redkloud                   A                              R
Miss Kelly                 A                              S
Montcalm                   A                              S
PC-50                      A        9                     R
US 3                       A        8                     S
Jose Beta                  A                              S
XAN-159                    A                              S
Pompadour Q1               A                              S
Pompadour D                A                              S
Pompadour U                A                              S

    Cultivar-breeding line         Coupling-phase RAPD and SCAR markers

Entry                    Origin    OBC06.300    OAG15.300    SOBC06.308

Pinto Olathe               MA          +            +            +
Pinto Apache               MA          +            +            +
Pinto Bill Z               MA          +            +            +
Pinto Burke                MA          +            +            +
Pinto Kodiak               MA          +            +            +
Pinto Topaz                MA          +            +            +
Pinto Chase                MA          -            -            -
Pinto Fiesta               MA          -            -            -
Pinto Othello              MA          -            -            -
Pinto P114                 MA          -            -            -
Pinto P650                 MA          -            -            -
Pinto UI-111               MA          +            +            +
Pinto US-5                 MA          -            -            -
Pinto US-14                MA          -            -            -
Pinto BelDak-RR-1          MA          +            +            +
Pinto BelDak-RR-2          MA          +            +            +
Pinto BelDakMi-RR-1        MA          +            +            +
Pinto BelDakMi-RR-2        MA          +            +            +
Pinto BelDakMi-RR-4        MA          -            -            -
Pinto BelDakMi-RR-5        MA          +            +            +
Pinto BelDakMi-RMR-10      MA          +            +            +
Pinto BelDakMi-RMR-14      MA          +            +            +
Pinto BelDakMi-RMR-18      MA          +            +            +
Pinto CO 12512             MA          +            +            +
Pinto CO 12515             MA          +            +            +
Pinto CO 12518             MA          +            +            +
Pinto CO 12555             MA          +            +            +
Pinto CO 12650             MA          +            +            +
Pinto CO 12652             MA          +            +            +
Pinto CO 12653             MA          +            +            +
Pinto CO 12655             MA          +            +            +
Pinto CO 12663             MA          +            +            +
Pinto CO 12783             MA          +            +            +
GN GN1140                  MA          -            -            -
GN Harris                  MA          -            -            -
GN Marquis                 MA          -            -            -
GN Valley                  MA          -            -            -
GN Nebr.#1                 MA          -            -            -
GN Nebr.#1 sel.27          MA          -            -            -
GN P#44                    MA          -            -            -
GN P#59                    MA          -            -            -
GN P#92                    MA          -            -            -
GN BelNeb-RR-1             MA          -            -            -
GN BelMiNeb-1112-1         MA          -            -            -
GN BelMiNeb-RMR-4          MA          +            +            +
GN BelMiNeb-RMR-7          MA          -            -            -
Black HT 7719              MA          -            -            -
Black A 55                 MA                       -            -
Navy Seafarer              MA          -            -            -
Navy Midland               MA          -            -            -
Navy BelMiDak-RR-8         MA          -            -            -
Aurora                     MA          -            -            -
Ecuador 299                MA          -            -            -
Mex 309                    MA          -            -            -
Viva                       MA          -            -            -
BAC 6                      MA          -            -            -
Chichara                   MA          -            -            -
Golden Gate Wax            A           +            +            +
Early Gallatin             A           +            +            +
Brown Beauty               A           +            +            +
Redkloud                   A           +            +            +
Miss Kelly                 A           +            +            +
Montcalm                   A           +            +            +
PC-50                      A           +            +            +
US 3                       A           -            -            -
Jose Beta                  A           -            -            -
XAN-159                    A           -            -            -
Pompadour Q1               A           -            -            -
Pompadour D                A           -            -            -
Pompadour U                A           -            -            -


ACKNOWLEDGMENTS

We acknowledge financial support from the Title XII Bean/ Cowpea CRSP (AID Contract No. DNA-1310-G-SS-6008-00). We also appreciate the constructive criticism of two reviewers, Drs. Don Lee and Steve Baenziger, Department of Agronomy and Horticulture, University of Nebraska-Lincoln, to improve the manuscript. We also thank technicians Janelle Fentom, Daniela O'Keefe, Lindsey Otto, and Lisa Sutton, University of Nebraska-Lincoln, for their assistance.

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S. O. Park, D. P. Coyne, J. R. Steadman, * K. M. Crosby, and M. A. Brick

S.O. Park and D.P. Coyne, Dep. of Agronomy & Horticulture, Univ. of Nebraska, Lincoln, NE 68583; J.R. Steadman, Dep. of Plant Pathology, Univ. of Nebraska, Lincoln, NE 68583; K.M. Crosby, Dep. of Horticulture, Texas A&M Univ., Weslaco, TX 78596; M.A. Brick, Dep. of Soil & Crop Sciences, Colorado State Univ., Fort Collins, CO 80523. This paper was published as paper no. 14205 Journal Series, Nebraska Agricultural Research Division. Research was conducted under Projects 20-036 and 20-042. Received 24 Sept. 2003. *Corresponding author (jsteadman1@unl.edu).
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Title Annotation:Genomics, Molecular Genetics & Biotechnology
Author:Park, S.O.; Coyne, D.P.; Steadman, J.R.; Crosby, K.M.; Brick, M.A.
Publication:Crop Science
Geographic Code:1USA
Date:Sep 1, 2004
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