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a USDA-ARS, 24106 N. Bunn Rd., Prosser, WA 99350
b USDA-ARS, Cornell University, Geneva, NY 14456
c Dep. of Agronomy and Range Science, University of California, 1 Shields Avenue, Davis, CA 95616-8515
Corresponding author (pmiklas{at}tricity.wsu.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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Abbreviations: cM, centimorgan • MAS, marker-assisted selection • QTL, quantitative trait locus or loci • RIL, recombinant inbred line
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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Germplasm lines and cultivars with upright architecture have become commonplace (Hosfield et al., 1995; Kelly et al., 1992a, 1992b), whereas development of cultivars which combine physiological resistance to white mold with upright architecture has progressed relatively slowly. Cumbersome screening methods, low heritability, and few available resistance sources have contributed to the lack of progress for enhancing physiological resistance. This situation may soon be alleviated, however, as the recently developed Bean White Mold Nursery (Steadman, 1995, 1997; Steadman et al., 1999) and novel, less cumbersome screening methods (Petzoldt and Dickson, 1996; Steadman, 1997; Kolkman and Kelly, 2000) are helping to identify, characterize, and select for physiological resistance to white mold in common bean (Miklas et al., 1998, 1999). The straw test (Petzoldt and Dickson, 1996) is a simple procedure for evaluating physiological resistance primarily within stem tissue. However, little is known about the inheritance of this trait.
The landrace cultivar G 122, has exhibited field resistance to white mold in the Bean White Mold Nursery (Steadman, 1997; Steadman et al., 1999) and elsewhere (Kmiecik and Nienhuis, 1998; Park et al., 1999a). The field resistance of G 122 likely results from physiological resistance. The breeding line A 55 expresses avoidance due to its upright architecture and narrow growth habit. Our objectives were to gain a better understanding of resistance to white mold in G 122 using QTL analysis, evaluate heritability of physiological resistance as detected by the straw test, and compare physiological resistance as detected by the straw test with field reaction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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Genetic Linkage Map
A linkage map for the A 55/G 122 RIL population was previously developed by Johnson (1997)(http://agronomy.ucdavis.edu/gepts/mapdata2.htm#framework; verified October 28, 2000). In a first step, a molecular linkage map consisting of 245 markers [222 AFLPs (amplified fragment length polymorphism), 10 RFLPs (restriction fragment length polymorphism), 3 RAPDs (random amplified polymorphic DNA), 2 SCARs (sequence characterized amplified region), 4 phenotypic markers, 3 isozymes, and 1 seed protein] was established in the A 55/G 122 population using Mapmaker/EXP 3.0 (Lander et al., 1987). A pairwise linkage analysis of the marker data, imposing a minimum LOD score of 3.0 and maximum distance of 30 centimorgans (cM), was used to establish the linkage groups. Three-point and multi-point log-likelihood thresholds (LOD) of 2.5 and 2.0, respectively, were used to order the markers within linkage groups with the Order and Ripple commands. Centimorgan distances between linked loci were based upon recombination fractions using the Kosambi (1944) mapping function.
Twelve linkage groups were identified, ten of which were anchored to the core map for common bean using previously linked markers (Freyre et al., 1998; http://agronomy.ucdavis.edu/gepts/mapdata2.htm#Coordination; verified October 28, 2000). The total map length was 853 cM. This is less than the map length of 1200 cM reported for common bean (Freyre et al., 1998), suggesting that more markers other than AFLPs, which tend to cluster around the centromere, are needed to obtain a more complete map. As a result of clustering among the AFLP markers, only 74 framework markers from the original 245 markers were chosen for QTL analysis. The markers were chosen so as to have as few missing data as possible and also have a regular 10-15 cM spacing. The markers for the framework map were composed of 60 AFLPs; 5 RFLPs; 3 phenotypic markers Asp, fin, and I; 2 RAPDs; 2 SCARs; 1 isozyme RBcS; and 1 seed protein Phs. The primers for the AFLP markers found to be linked to a white mold resistance QTL were A05 (5'-G ACT GCG TAC CAA TTC ACA-3'), A12 (5'-G ACT GCG TAC CAA TTC AGT-3'), P9 (5'-G ACG ATG AGT CCT GAG TAA AGA-3'), P10 (5'-G ACG ATG AGT CCT GAG TAA AGC-3'), and P11 (5'-G ACG ATG AGT CCT GAG TAA AGG-3'). A combination of restriction enzymes, EcoRI and MseI, was used to digest the genomic DNA prior to PCR amplification.
Greenhouse Straw Tests
The straw test described by Petzoldt and Dickson (1996) was used to screen the RIL population, parents, and checks in two separate greenhouse environments. Straw Test 1 (planted 14 Oct. 1997) and Straw Test 2 (planted 24 Feb. 1998) consisted of five and six replications, respectively. An individual plant of each line represented a replicate and the replications were randomized in complete blocks. The parents were inadvertently excluded from the first test. For both tests the greenhouse environment was maintained at 20°C/night to 28°C/day with a 14-h daylength provided by sunlight and supplemental artificial lighting. Plants were watered and fertilized for normal growth. The S. sclerotiorum culture T001.1, hyphal-tip isolated from a sclerotia collected from ‘Newport’ navy bean in Quincy, WA, in 1996, was the source of inoculum. Inoculation took place approximately 28 d after planting following the procedure of Petzoldt and Dickson (1996). Briefly, the growing tip of the main stem of a single plant, sown in a 10- to 15-cm-diameter pot containing a soilless potting mix (Sunshine No. 1; Fison Hort., Vancouver, BC), was discarded and a plastic straw containing an agar plug of mycelium of the pathogen was fitted over the intact cut stem of the whole plant. The entire petri plate of growing mycelium was used as inocula soon after mycelium had grown from the center to the periphery of the plate. Eight days after inoculation, the white mold reaction was scored from 1 to 9, where 1 = no symptoms, 2 = invasion of the stem past the site of inoculation but not to the first node, 3 = invasion of the stem to the first node, 4 = invasion of the internode slightly past the first node, 5 = invasion to the middle of the internode, 6 = invasion to the second node, 7 = invasion slightly past the 2nd node, 8 = invasion to the middle of the second internode and beyond, and 9 = total plant collapse (Petzoldt and Dickson, 1996; Miklas et al., 1999).
Field Test
The field plot at the USDA-ARS Cropping Systems Research Farm at Patterson, WA, has a history of S. sclerotiorum disease in potato (Solanum tuberosum L.) and pea (Pisum sativum L.). Nevertheless, 120 kg ha-1 of sclerotia mixed with seed tailings obtained from a local bean elevator (Central Bean, Quincy, WA) were incorporated across the plot in October 1998. The soil is a Quincy sandy loam (mixed, mesic Typic Torripsamments). The RILs and parents were planted 24 May 1999 in a randomized complete-block design with three replications. A plot consisted of four rows, 3 m long, and spaced 0.56 m apart. Planting density was 234 848 seeds ha-1. To promote white mold disease, approximately 6.3 mm of water was applied on a daily basis from the onset of flowering to late pod-fill by overhead center-pivot irrigation. To maintain vigorous plant growth, nitrogen was foliar applied at a rate of 22 kg ha-1 on 14 June, 2 July, 22 July, and 11 August by chemigation.
Disease reaction within the middle two rows was scored from 1 to 9 on the basis of combined incidence and severity of infection at physiological maturity (8 September), where 1 = no diseased plants; 2 = 1 to 20% diseased plants and/or 1 to 5% infected tissue; 3 = 20 to 30% diseased plants and/or 5 to 10% infected tissue; 4 = 30 to 40% diseased plants and/or 10 to 20% infected tissue; 5 = 40 to 50% diseased plants and/or 20 to 30% infected tissue; 6 = 50 to 60% diseased plants and/or 30 to 40% infected tissue; 7 = 60 to 70% diseased plants and/or 40 to 50% infected tissue; 8 = 70 to 80% infected plants and/or 50 to 60% infected tissue; and 9 = 80 to 100% diseased plants and/or 60 to 100% infected tissue. Traits measured at mid-pod fill (9 August) included canopy height (cm) and canopy porosity (Deshpande, 1992) scored from 1 to 5, where 1 = an open canopy with the soil surface between rows completely visible and 5 = completely closed canopy over the furrow with no soil visible.
Inheritance and QTL Analysis
Analysis of variance for each trait was performed by PROC GLM (SAS, 1987). A narrow-sense heritability estimate for disease score for each test was computed with variance components on an F8-derived line-mean basis (Hallauer and Miranda, 1981). This h2 approximates a narrow-sense estimate because coefficients for nonadditive genetic components in the expectation of genetic variance are near zero for F8-derived lines. Exact 90% confidence intervals were calculated for h2 according to the procedures of Knapp et al. (1985). Error mean squares for the separate analyses of variance for the two straw tests were homogeneous based on Bartlett's test (Steel and Torrie, 1980); therefore, a combined analysis of variance was conducted to obtain h2 for disease score across straw tests. Frequency distributions of the RIL means for disease score were tested for normality by the Shapiro and Wilk test statistic W (PROC Univariate, SAS, 1987). A probability of P < 0.001 was used to indicate lack of fit.
Simple interval mapping performed by MQTL (Tinker and Mather, 1995) was used to detect QTL conditioning physiological resistance in the straw tests, field resistance, and agronomic traits associated with disease avoidance, along the framework map of 74 markers (Johnson, 1997). We performed permutation analyses (1000 permutations) of the disease score data sets (Doerge and Rebai, 1996) from the straw tests and field in order to identify a significance threshold of the test statistic for a QTL based upon a 10% experiment-wise error rate. The threshold value thus determined for the test statistic was 12.4 for the straw tests and 12.8 for the field which when multiplied by 0.22 equates to LOD scores of 2.7 and 2.8 respectively. Significant QTL were then declared if the test statistic calculated by MQTL was greater than 12.4 (Straw tests) or 12.8 (field). The R2 values for describing the phenotypic variation explained by a significant QTL were calculated as (variance explained) / (total variance). The effects of QTL and agronomic traits on disease score in the field was modeled by multiple regression (PROC STEPWISE, SAS, 1987). A significance level of 0.15 was required for a trait to be included in the model.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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2p) is biased upward because of the presence of 2ge in the numerator. Both parents expressed field resistance, however, less disease for A 55 (2.7) than G 122 (3.8) indicated avoidance was expressed (Table 1). This result provides further support of the extreme importance that disease avoidance has on reducing white mold disease in the field. Physiological resistance was also expressed. Field reaction to white mold is confounded by physiological resistance and avoidance making it difficult to separate the contribution of each to overall resistance. In this study QTL mapping in the A 55/G 122 population enabled some separation of the different mechanisms.
The same B7 QTL derived from G 122 in the general area of the Phs locus conditioning physiological resistance in the straw test was expressed in the field (Table 3 and Fig. 2). Small population size, which renders QTL location less precise, is likely responsible for the slight shift in QTL location away from the Phs locus and toward the A5/P9 T AFLP marker in the field test. Positive correlations (Table 2) between disease scores from the field and straw tests support the presence of a common QTL conditioning physiological resistance in the field and greenhouse.
Given the major effect of the QTL detected on B7, it will be worthwhile to determine the potential for Phs and associated AFLP markers to indirectly select for physiological resistance derived from G 122 in different segregating populations. The potential use of Phs for marker-assisted selection (MAS) of the QTL would be restricted initially to certain snap bean lines and dry beans of Middle American origin that possess S phaseolin because G 122 has the T phaseolin. Caution in the interpretation of the QTL results should be exercised, however, as the small size of the A 55/G 122 RIL population leads to an overestimate of the actual effect of the resistance gene and to a reduction in the precision of the location of the gene (Melchinger et al., 1998). Thus, to achieve efficient MAS, it may be necessary to identify markers more tightly linked or flanking the QTL in larger populations. The identification of a major QTL conditioning physiological resistance in the straw test located on a different linkage group, B8, and from a different source, landrace Pompadour Checa 50 (Park et al., 1999b), provides an opportunity for examining the usefulness of combining a different source of physiological resistance to white mold with that of G 122.
It is noteworthy, that four additional disease resistance genes have mapped to the same region of B7 as the QTL for physiological resistance to white mold. The four loci are major QTL for resistance to common bacterial blight, bean golden mosaic, and ashy stem blight caused by Macrophomina phaseolina (Tassi) Goid. (Nodari et al., 1993; Miklas et al., 2000), and a gene for resistance to Colletotrichum lindemuthianum (Sacc. et Magn.) Scrib., causal agent of anthracnose (Geffroy, 1997). As has been observed before in other species (Ashfield et al., 1998; Witsenboer et al., 1995) and beans (Geffroy et al., 1998 and 1999; Miklas et al., 2000; Stavely, 1984), disease resistance genes often occur in clusters.
A second QTL (18%) conditioning resistance to white mold in the field was identified on linkage group B1 near the fin locus for determinate bush growth habit (Table 3). A 55 has the genotype Fin/Fin conditioning indeterminancy and G 122 has fin/fin conditioning determinancy. This QTL could be associated with avoidance to white mold because a QTL (34%) for porous canopy was located in the same region. Note that open canopy was associated with less disease when estimated on an individual plot basis (r = 0.27, 189 df, P < 0.01), but not when analyzed on a mean basis (Table 2). There was no interaction between the QTL for field resistance on B1 and B7, nor did they appear to have a completely additive effect, as together they explained only 38% of the variation for disease score. The lack of an interaction or completely additive effect provides some additional support that the two identified QTL likely have independent effects, ie., avoidance for the B1 QTL and physiological resistance for the B7 QTL.
The multiple regression model of field reaction to white mold [y = 6.93 - 0.08(height) + 0.99(B7 QTL) + 1.08(fin)] provides additional support for the importance of both physiological and avoidance factors on the expression of field resistance in the A 55/G 122 population. The B7 QTL (A05/P09 T) had the largest effect (21%) as measured by the partial R2 contribution to the model, followed by plant height (8.7%), and the B1 QTL or fin gene (8.4%). Thus, the quantitative inheritance of field resistance to white mold in the A 55/G 122 population is due in part to both physiological resistance and avoidance mechanisms. The size of the mapping population only enabled detection of QTL which accounted for more than 10% of the phenotypic variation for disease score. Thus, other QTL with a minor influence on resistance could exist in the population.
Oddly, lines with determinate bush growth habit (fin) had less disease than the lines with indeterminate vine growth habit (Fin), even though A 55 had less disease than G 122 in the field. This suggests that the narrow upright Type II plant profile of A 55 did not result in disease avoidance in the RIL population. This is substantiated by the population mean for plant height (52.4) being less than the parental means and mean canopy porosity (2.4) similar to the less porous parent G 122. Regardless of the growth habit present the importance of increased plant height was associated with less disease (r = -0.37) in the field.
The loss of parental ideotype attests to the reported difficulty of obtaining useful germplasm from wide crosses between the Middle American and Andean gene pools (Singh, 1999). Many of the determinate bush RILs seemed to condition avoidance due to a lack of canopy closure and less biomass. Thus, the positive effect of the fin gene (bush growth habit) on avoidance in the A 55/G 122 population was primarily an artefact of poor vigor generated by the wide cross. The fact that less disease was partly associated with avoidance resulting from poor vigor attests to the need for simultaneous selection for high yield and resistance to white mold.
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SUMMARY AND CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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The landrace cultivar G 122 provides breeders with a heritable source of physiological resistance to white mold disease in common bean. The development of such resistance should follow a strategy whereby selection for physiological resistance is performed first among F3 or later generation lines using the straw test. Secondly, to combine this resistance with avoidance, the most resistant lines in the straw test would then be tested in a white mold field nursery. Only the highest yielding and least diseased lines would be advanced for additional cycles of testing and crossing. Thirdly, the transferred resistance should eventually be combined with other resistance sources, especially those known to possess different mechanisms and QTL for resistance, using recurrent selection or MAS, to obtain bean cultivars with enhanced resistance to white mold disease.
Received for publication January 12, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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