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a PO Box 839, Williamsburg, IA 52361
b Dep. of Plant Sciences, North Dakota State Univ., Fargo, ND 58105
c Dep. of Crop and Soil Sciences, Michigan State Univ., E. Lansing, MI 48824
Corresponding author (kellyj{at}msu.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: BGMV, bean golden mosaic virus • bp, base pair • CBB, common bacterial blight • cM, centimorgan • DAP, days after planting • DTF, days to flower • FSP, Fusarium solani f. sp. phaseoli • IF, Isles/FR266 • MAS, marker-assisted selection • MF, Montcalm/FR266 • PI, Presque Isle county, MI • PCR, polymerase chain reaction • PM, Perham, MN • RAPD, random amplified polymorphic DNA • RCBD, randomized complete block design • RILs, recombinant inbred lines • QTL, quantitative trait loci
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Improvement of root rot resistance, especially in large-seeded and snap bean types, has been limited, in spite of considerable research efforts to elucidate the genetic control of resistance in bean to FSP. Previous approaches employed to elucidate the inheritance of resistance to FSP involved experimental designs more appropriate for the analysis of qualitative rather than for quantitative traits (Smith and Houston, 1960; Wallace and Wilkinson, 1965; Hassan et al., 1971). In addition, scoring individual plants can be problematic for complexly inherited traits since an average score for a particular genotype is preferred for traits strongly influenced by environmental factors. Lack of complete resistance to FSP in P. vulgaris, and observable differences in levels of susceptibility, support the proposed quantitative inheritance of this trait (Baggett et al., 1965). The strong environmental influence on disease incidence and severity ratings for FSP provides additional evidence for the complex inheritance of resistance (Miller and Burke, 1985). These observations emphasize the need to analyze root rot resistance as a quantitative trait.
While the control of environmental variation through replicated field trials is important for analyzing resistance to root rot, the actual scoring method is equally significant. Root damage has been implicated as a more accurate indicator of yield reductions caused by FSP than the conventional rating of hypocotyl lesions (Beebe et al., 1981; Burke and Barker, 1966). A method for evaluation that includes assessment of lateral root infection rather than hypocotyl reaction will provide more relevant results. Another confounding problem with previous genetic studies was the use of wide, inter-gene pool crosses to study inheritance of resistance (Smith and Houston, 1960; Bravo et al., 1969; Hassan et al., 1971). While recombination between Andean and Middle American gene pools occurs readily, hybrid lethality can result (Koinange and Gepts, 1992). Skewed segregation as a result of this phenomenon is common and may lead to misinterpretation in inheritance studies.
FR266 is a determinate root rot resistant line possessing a large root system, white seed, and snap bean-like pods (Silbernagel, 1987). Resistance was transferred from the small, viny, black seeded, Middle American, weedy bean landrace from Mexico, N203 (PI 203958), identified by plant collector and explorer, Oliver Norvell, as resistant to root rot (Wallace and Wilkinson, 1966). The Andean breeding line FR266 provided an opportunity to study inheritance patterns within the same gene pool, while offering the chance to improve resistance in the most susceptible bean market classes.
Molecular markers may prove valuable for breeding improved FSP resistance in bean. RAPD markers associated with QTL controlling resistance to common bacterial blight [CBB; Xanthomonas campestris pv. phaseoli (Smith, 1897) Dye 1978], and halo blight [Pseudomonas syringae pv. phaseolicola (Burkholder, 1926) Young, Dye & Wilkie, 1978] have been identified (Ariyarathne et al., 1999; Jung et al., 1999; Miklas et al., 1996) in bean. Marker-assisted selection (MAS), used to select indirectly for resistant genotypes, may facilitate improvement of disease resistance for traits, like root rot resistance, where field selection is laborious and destructive. While the practical application of MAS for quantitative traits has yet to be realized, many studies recognize its potential to facilitate improved disease resistance controlled by quantitative traits (Pilet et al., 1998; Mangin et al., 1999; Schechert et al., 1999; Miklas et al., 1998; Faris et al., 1999; Lubberstedt et al., 1998). QTL-marker associations also may provide a basis for a greater understanding of quantitative disease resistance through the identification of loci that influence resistance to more than one disease (Ariyarathne et al., 1999). The objective of this study was to characterize the inheritance of resistance to FSP by means of recombinant inbred line (RIL) populations and to identify significant QTL-marker associations for resistance to root rot in bean.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The IF population was constructed as a means to verify marker associations in a population other than the one in which the marker was identified. This strategy involved the identification of markers in the MF RIL population and subsequent confirmation of these associations based on data generated from IF evaluation.
Field Tests
Field trials were conducted in both Presque Isle County, Michigan (PI) and Perham, MN, (PM) in 1997 and 1998. Selected fields were previously identified as infested with FSP. All field experiments were planted in a randomized complete block design (RCBD). Standard agronomic practices were applied to ensure good crop growth and development. A combination of pre-emergence herbicides and both mechanical and hand cultivation were used to control weeds. Seed was treated with a three-way combination of a commercial fungicide, bacteriocide and insecticide. Single-row plots planted with 10 seeds each were 1.2 m long and 0.76 m wide. Five plants per plot were uprooted with a shovel, taking care not to disturb the main portion of the root system. Roots were cleaned of debris and rated on a scale from 1 to 7 (1 = resistant, 7 = susceptible; Schneider and Kelly, 2000).
In addition to the root rot evaluation experiments, all RILs for MF were increased in four- and two-row unreplicated plots, respectively, in Ingham County, Michigan, in 1998. Data for days to flower (DTF) and days to maturity were recorded in this experiment.
Greenhouse Tests
Both RIL populations were evaluated for root rot resistance in the greenhouse using a perlite-based protocol (Schneider and Kelly, 2000). Bean seed were germinated in 72-well flats in perlite. Seedlings were inoculated with pathogenic FSP strains isolated from Presque Isle (Hawks 2b and F.s. 12) and Huron (Huron 2a and 2b) counties in Michigan (Schneider and Kelly, 2000). A 1 x 105 spore mL-1 solution of FSP macroconidia diluted in deionized water was applied with a hand pump 4-L sprayer, 10 d after planting (DAP). Fourteen days after inoculation seedlings were uprooted, washed of excess perlite and rated on a scale from 1 to 7.
MF Population
Field trials for MF were planted on 4 June 1998 at PI and on 21 May 1998 at PM. Seventy-nine F4:6 RILs, two parents, and 10 checks were planted in PI in an RCBD with four replications. Plots were rated 78 DAP. Seventy-eight F4:6 RILs, two parents and four checks were planted in PM in an RCBD with three replications. Plots were rated 74 DAP. Only one rating, recorded during late pod-fill, was taken for these 1998 MF experiments due to problems with plant emergence. Three greenhouse screenings were conducted with F4:5 (MFGH1) and F4:7 (MFGH2, MFGH3) MF populations on three plants per genotypic entry using an RCBD with four replications. Seedlings in MFGH1, MFGH2 and MFGH3 were inoculated with F.s. 12, Huron 2a and Hawks 2b isolates, respectively. The genotypes included in these screenings were the same as those used in the 1998 PI trial.
IF Population
A recombinant-inbred population derived from a cross between Isles and FR266 (IF) was advanced by single seed descent in the greenhouse. DNA from F2 individuals of IF was extracted and markers identified in MF were used to confirm the effectiveness of early generation selection based on greenhouse screening of IF. A single unreplicated greenhouse evaluation consisting of six plants each of 68 F4:5 RILs, parents and six checks was conducted to verify associations. Plants were inoculated with isolate Hawks 2b and was limited to six plants per genotype because of a lack of adequate seed quantities.
Statistical Analysis
All experiments conducted in the field and greenhouse involving MF were analyzed as one-way analyses of variance (ANOVA) using PROC GLM and PROC MIXED (SAS Institute, 1994). Root rot ratings from the five plants scored from each plot were averaged to give a plot mean that was subsequently used for ANOVAs. Because of uneven plant numbers per plot, analysis of experiments conducted in PI and PM in 1998 included a subsampling error. Heritability estimates were calculated on an entry mean basis for all greenhouse and field trials according to Hallauer and Miranda (1981). Pearson rank correlation coefficients were calculated by PROC CORR to compare means of root rot ratings for RILs within MF for all greenhouse and field evaluations conducted for this population (SAS Institute, 1994).
Marker Analysis
DNA was extracted from MF and IF by either a mini-extraction protocol described by Schneider et al. (1997) or a larger quantity DNA extraction method described by Kisha et al. (1997). Tissue from F2 progenitors of corresponding F4:5 RILs in IF, respectively, was sampled for DNA extraction. Tissue from six plants of each of the F4:6 MF RIL was bulked and extracted.
RAPDs were generated by the protocol outlined by Schneider et al. (1997) with slight modification. PCR products and repeatability was enhanced by doubling the amount of enzyme used in each reaction. RAPDs greater than 1200 bp were amplified by GIBCO BRL brand Taq polymerase (Life Technologies, MD) and RAPDs less than 1200 bp were amplified by AmpliTaq DNA polymerase, Stoffel Fragment (Perkin Elmer, CT). Seven hundred random 10-mer primers (Operon Technology, CA) were used to amplify random regions of the genome. DNA was amplified with a Perkin Elmer Cetus DNA Thermal Cycler 480 (Perkin Elmer, Cetus, Norwalk, CT) using the following cycles: 3 cycles of 1 min. at 94°C, 1 min. at 35°C, and 2 min. at 72°C, 34 cycles of 1 min. at 94°C, 1 min. at 40°C, and 2 min. at 72°C (final step extended by 1 sec for each of the 34 cycles), and a final extension cycle of 5 min. at 72°C (Haley et al., 1994). Approximately 20 µL of amplified DNA from each sample was run on a 1.4% (w/v) agarose gel containing ethidium bromide (0.5 µg mL-1), 40 mM Tris-acetate, and 1 mM EDTA. DNA was viewed under ultraviolet light and photographed for permanent record.
Primers were screened against Montcalm and FR266 parents to identify polymorphisms that would subsequently be utilized in the population analyses. A method of selective genotyping used by Miklas et al. (1996), was employed to facilitate identifying markers associated with QTL controlling FSP resistance in MF. DNA from the five most resistant RILs of MF, based on data from MFGH2, was pooled to create a resistant bulk (R). Likewise, DNA from the five most susceptible RILs was pooled to create a susceptible bulk (S). RAPD markers polymorphic between parents and the R and S bulks were subsequently screened against individual members of the bulk. If the RAPD continued to cosegregate according to R and S classifications of the individuals used to make the bulks, the primer was screened against the entire population. RAPDs reported on the core P. vulgaris linkage map (Freyre et al., 1998) also were used for screening MF, although many were not polymorphic between parents and, thus, uninformative.
Significant associations (P < 0.05) between FSP resistance and marker genotype were determined by one-way ANOVAs using PROC GLM (SAS Institute, 1994). Multiple regression analyses using combinations of significant markers also were performed by PROC GLM. RAPDs that did not exhibit a 1:1 segregation based on chi-square results were discarded. Linkage relationships among the remaining markers were determined by MAPMAKER/EXP group command with minimum LOD of 4.0 and maximum distance of 50 cM (Lincoln et al., 1987).
Significant markers identified in MF with P < 0.05 were subsequently screened against IF to verify associations unless the RAPD was not polymorphic between Isles and FR266. Some primers, like G6, when screened against the parental genotypes of IF, generated additional polymorphisms from those identified in MF. These additional RAPDs were scored in IF.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Multiple regression analyses using combinations of significant markers and their interactions revealed that epistatic interactions were not significant. Up to 14 and 29% of the phenotypic variation for greenhouse and field ratings, respectively, was explained by a set of markers that included G32000, G17900, U121500 and P7700. Interestingly, this same set of markers explained 24% of the variation for DTF. RAPDs U121500 and G17900 were eliminated from the aforementioned multiple regression analysis because both were strongly associated with DTF. The remaining two markers G32000 and P7700 accounted for 7 to 13% of the phenotypic variation for root rot ratings in both field and greenhouse with no significant association observed with DTF.
Primers G3, G6, and P7 were screened against the 68 F2 individuals from IF. RAPDs G6300 and G3800 were linked at 7.6 cM in IF but G6300 was not a polymorphic locus in MF. G32000 was not polymorphic in IF, whereas G61100 and G3800 were not associated with F4:5 greenhouse root rot scores. P7700 was significantly associated (P < 0.05) with root rot ratings for this population, confirming previously reported associations for this marker in MF populations.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The disease reaction of an FSP resistant genotype resembles that of a susceptible genotype in symptomology except that disease progress in the resistant genotype is substantially reduced. An incompatible reaction involving the hypersensitive response is not observed for resistant genotypes inoculated with this pathogen. Under severe disease pressure, resistance can be overcome, as evidenced by root rot ratings greater than 4.0 for the resistant parent, FR266 (Table 1). Susceptible genotypes, however, scored consistently higher than the resistant parents, supporting the conclusion that rate of progress of disease is substantially reduced in the resistant genotypes and that environmental factors play a large role in disease severity (Table 1).
In the current study, several regions and independent markers were identified which significantly associated with field and greenhouse root rot ratings, but none explained over 15% of the phenotypic variation for this trait (Table 3). In multiple regression analyses, only 29% of the phenotypic variation could be accounted for by a subset of four markers. QTL analysis for resistance to other bean diseases, such as bean golden mosaic virus (BGMV) and CBB, revealed four and five QTL for BGMV and CBB, respectively (Miklas et al., 1996). Soilborne pathogens have a complex life cycle that is strongly influenced by environmental factors. Only three of the eight RAPDs significantly associated with Macrophomina phaseolina (Tassi) Goidanich resistance in bean were consistently significant across two locations (Miklas et al., 1998). The large amount of environmental variation observed for diseases caused by soilborne pathogens makes quantification difficult and could explain why QTL associated with these traits are more difficult to resolve.
Markers that were significantly associated with field root rot ratings in this study were generally not significantly associated with greenhouse root rot ratings and vice versa. Furthermore, those RAPDs associated with field ratings tended to be associated with DTF (Table 3). These results suggest independent mechanisms involved in resistance to FSP between natural and artificial inoculations and that natural infections may reflect differences in phenology. Data from greenhouse experiments were generally significantly and positively associated with field trials for MF except for the nonsignificant but positive correlation between MFGH2 and 1998 PI field trial (Table 2). Data from repeated greenhouse screenings of MF were positively correlated among experiments and reflect segregation of physiological resistance in this population (Table 2). The observed trend that the same QTL associated with greenhouse ratings were not associated with field experiments and vice versa is not surprising. Genetic variation for physiological resistance present in MF may have been masked by strong environmental factors present in field trials such that QTL associated in greenhouse trials could not be resolved from field data. Furthermore, environmental effects influencing disease development present in field trials were absent in greenhouse trials and QTL associated with these factors were, therefore, not identified.
An example of an environmental factor present in field trials and not in greenhouse is DTF. Greenhouse plants were rated for disease severity before flowering but field trial ratings were conducted post-bloom. The significant negative relationship between DTF and field root rot ratings could also correspond to differences in developmental stages at the time of rating (Table 2). All RILs were rated at the same time regardless of development. Thus, those genotypes that were more mature also had more disease. This association between maturity and disease has been observed for other pathogens (Miklas et al., 1996; Pilet et al., 1998; Schechert et al., 1999) and was reported previously for FSP (Abawi, 1989) in bean. A negative association between FSP resistance and DTF is not unexpected, however, especially when considering RILs in MF population, with determinate growth habit. In these genotypes, vegetative growth ceases at flowering and the plant partitions its resources to the developing seed, resulting in less available energy for secondary processes like host defense. Furthermore, subsequent root decay may tend to favor the pathogen through the release of nitrogenous compounds that can stimulate aggressiveness of the fungus (Toussoun, 1970). Considering the negative correlation between DTF and root rot resistance, breeders intending to improve root rot resistance would benefit from selection methods like greenhouse assays and MAS that avoid concomitantly increasing DTF and associated late maturity.
Two linkage groups (LG2 and LG5) for which all markers were consistently associated with either greenhouse or field ratings were identified in MF. Markers P7700, P101600, and G61100 on LG2 were all significantly associated with root rot in at least one of the greenhouse trials conducted, and P700 also was significantly associated with root rot ratings in the 1998 PM trial and combined analysis (Table 3). Markers on LG5 were all positively and significantly associated with field root rot ratings but not greenhouse ratings. P700 and G61100 from LG2 are located on B2 and span a region that encompasses the PvPR2 locus, suggesting a role for this PR protein in root rot resistance. PvPR2 and its counterpart PvPR1 are low molecular weight acidic proteins induced during fungal elicitation (Walter et al., 1990). These bean PR proteins share similarities with PR proteins in crops such as pea (Pisum sativum L.), potato (Solanum tuberosum L.), and parsley [Petroselinum crispum (Miller) Nyman ex A.W. Hill]. The role of PvPR proteins in FSP resistance is further confirmed by the significant association observed between S800 from LG7 and I181800 of LG3 which map to B3 in the region of PvPR1 gene (Table 3). Differences in PvPR gene arrangements were detected between anthracnose [Colletotrichum lindemuthianum (Sacc. & Magnus) Lams.-Scrib.] resistant and susceptible bean genotypes indicating that polymorphism between PvPR as well as other defense response-related genes may contribute to our understanding of quantitative resistance (Walter et al., 1990). QTL associated with resistance to the late blight fungus [Phytophthora infestans (Mont.) de Bary] of potato also have been reported to associate with two potato PR proteins (Gephardt et al., 1991). To capitalize on the assumption that defense proteins may be associated with quantitative resistance, a method of candidate gene analysis, whereby genes known to be involved in host defense response are used as markers to identify potential QTL associated with disease resistance, has been applied in other crops (Goldman et al., 1993; Causse et al., 1995; Byrne et al., 1996; Faris et al., 1999) and could be employed to improve root rot resistance in bean.
RAPDs G32000 and P7700 were used in a multiple regression analysis and demonstrated significant associations with all greenhouse and field trials explaining between 7 to 19% of the genetic variation for root rot scores in MF. Selection based on these markers will be useful but not sufficient to improve root rot resistance. Neither of these markers was associated significantly with DTF. Analysis of F4:5 RILs from IF population indicated that P7700 was significantly associated with greenhouse root rot scores in this population supporting the association observed in MF. G32000 was not polymorphic in this population, whereas G61100 and G3800 were not significantly associated with root rot ratings. This is not surprising since G3800 was only associated with field ratings and not with greenhouse ratings. These results indicate that P7700 will be a useful marker for early generation selection for FSP resistance when FR266 is used as a resistance source.
Marker-assisted selection using P7700 and G32000, combined with later generation conventional selection, has the potential to enhance progress towards incorporating root rot resistance in dark red kidney bean genotypes. Introgressing FSP resistance into large-seeded kidney germplasm will necessarily involve utilizing resistance sources from other market classes. As such, agronomically acceptable dark red kidney genotypes possessing root rot resistance will not be achieved through single-cross breeding approaches. Systems involving recurrent selection and backcross breeding are the preferred alternatives to improve this trait. Utilizing markers and greenhouse seedling evaluations to facilitate selection provides an opportunity for breeders to make educated decisions about which progeny should be intermated or backcrossed before crosses are performed. This additional information can save valuable time and resources. Alternatively, RAPD markers associated with QTL controlling FSP resistance can be used to select progeny possessing complementary QTL for intermating. A genotype possessing the P7700 allele can be recombined with another genotype possessing the G32000 allele with the ultimate goal of combining these two QTL for resistance into a single genotype that will possess higher levels of resistance than either genotype alone.
An overemphasis on the improvement of quality traits in the kidney and snap bean market classes may be responsible for the intense susceptibility to FSP in these seed and pod types as compared to small-seeded types. These factors are compounded by the observation that genetic diversity, in general, is lacking within the cultivated Andean bean germplasm (Becerra Velasquez and Gepts, 1994; Sonnante et al., 1994). Alternatively, unknown differences in host defense responses between gene pools may provide an explanation for the high degree of susceptibility to root rot observed in large-seeded germplasm of P. vulgaris. Despite the differences between resistance levels in the two gene pools, mechanisms associated with host defense responses appear to be involved in resistance to FSP. Selection, either direct or indirect, towards enhancing these traits should allow for rapid improvement of resistance to Fusarium root rot in Andean bean genotypes.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication March 17, 2000.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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