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Crop Science 41:1494-1498 (2001)
© 2001 Crop Science Society of America

CROP BREEDING, GENETICS & CYTOLOGY

Detection and Mapping of a Major Locus for Fusarium Wilt Resistance in Common Bean

A. L. Falla, P. F. Byrne*,a, G. Jungb, D. P. Coynec, M. A. Bricka and H. F. Schwartzd

a Dep. of Soil and Crop Sciences, Colorado State Univ., Ft. Collins, CO 80523
b Dep. of Plant Pathology, Univ. of Wisconsin, Madison, WI 53706
c Dep. of Horticulture, Univ. of Nebraska, Lincoln, NE 68583
d Dep. of Bioagricultural Sciences and Pest Management, Colorado State Univ., Ft. Collins, CO 80523

* Corresponding author (pbyrne{at}lamar.colostate.edu)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Fusarium oxysporum Schlectend. Fr. f. sp. phaseoli J.B. Kendrick and W.C. Snyder (FOP) is a vascular pathogen that causes Fusarium wilt in common bean (Phaseolus vulgaris L.). This disease is an increasing problem in the western U.S., and exploitation of genetic resistance is considered the most feasible control method. The objective of this study was to detect quantitative trait loci (QTL) for Fusarium wilt resistance in a population derived from an interracial cross between FOP-susceptible Belneb RR-1 (race Durango) x FOP-resistant A55 (race Mesoamerica). Seventy-six F6-derived recombinant inbred lines (RILs) were screened for disease severity in greenhouse inoculations and rated on a scale of 1 (resistant) to 9 (susceptible). The phenotypic data were compared to existing random amplified polymorphic DNA (RAPD) marker data using single-factor analysis of variance. Marker U20.750 on linkage group (LG) 10 accounted for 63.5% of the phenotypic variance for this trait. Lines exhibiting the A55 banding pattern at this locus had disease severity scores that averaged 3.6 points lower than lines with the Belneb RR-1 pattern. Two additional markers, AD4.450 on LG 3 and K10.700 on LG 11, were significant (P < 0.01) in single-factor analysis of variance, but only marker U20.750 on LG 10 remained significant when composite interval mapping (CIM) was conducted. The tight linkage between the putative QTL and U20.750, as indicated by CIM, makes this marker a promising candidate for conversion to a sequence-characterized amplified region (SCAR) for use in marker-assisted selection in Fusarium wilt resistant common bean cultivar development.

Abbreviations: CIM, composite interval mapping • DSI, disease severity index • FOP, Fusarium oxysporum f. sp. phaseoli • LG, linkage group • LOD, log of the odds • RAPD, random amplified polymorphic DNA • RIL, recombinant inbred line • SCAR, sequence-characterized amplified region • QTL, quantitative trait locus (loci)


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
FUSARIUM WILT is a vascular disease of common bean. This fungus has been detected in bean-growing regions throughout the world, and is an economically significant problem in Latin America, Africa, and the western United States (Pastor-Corrales and Abawi, 1987; Buruchara and Camacho, 2000; Salgado et al., 1995). Infected plants display symptoms of yellowing, wilting, and necrosis of leaf and stem tissue, which often results in hastened maturity, decreased seed size, and yield loss (Schwartz et al., 1996). In the High Plains region of the U.S., yield losses in fields affected with Fusarium wilt are estimated at 10 to 30% (Salgado et al., 1995). Fusarium wilt is difficult to control due to formation of chlamydospores that remain viable in soil for long periods of time. Chemical, cultural, and biocontrol treatments cannot effectively limit this disease, making genetic resistance the most viable control measure.

Genetic resistance to FOP race 4 has been identified in germplasm from common bean races Durango and Mesoamerica of the Middle American gene pool (Velasquez-Valle et al., 1997). In some populations of Durango, inheritance of resistance to this isolate is controlled by a single dominant gene, designated Fop4 (Salgado et al., 1995; Cross et al., 2000). However, attempts to identify a resistance locus using bulked segregant analysis have been unsuccessful in two Durango populations (Cross, 1998). In Mesoamerica, there are indications of polygenic resistance to Fusarium wilt (Salgado et al., 1995; Cross et al., 2000).

Resistance to Fusarium wilt has also been identified in the Andean gene pool of common bean (Ribeiro and Hagedorn, 1979; Velasquez-Valle et al., 1997). Ribeiro and Hagedorn (1979) identified dominant single gene resistance to FOP race 1 and incompletely-dominant single gene resistance to race 2 in snap bean populations. They considered each of these to be due to separate genes, and designated them Fop1 and Fop2, respectively.

Molecular marker analysis and QTL mapping techniques have been useful tools for locating and analyzing loci that confer resistance to plant pathogens (Young, 1996). Markers for major QTLs may be used to accelerate the process of resistant cultivar development through marker-assisted selection (Kelly and Miklas, 1999). In the case of Fusarium wilt, disease screening is problematic because of nonuniform distribution of infection in the field, labor-intensive greenhouse inoculations, and the strong influence of environmental conditions on disease development. These factors make Fusarium wilt resistance loci ideal candidates for marker-assisted selection if major QTLs are detected.

Quantitative trait loci have been reported for resistance to several of the major pathogens of common bean (Kelly and Miklas, 1999; Ariyarathne et al., 1999). No QTLs or major genes for Fusarium wilt resistance have been mapped in common bean. However, both quantitative and qualitative resistance to other formae speciales of F. oxysporum have been identified and mapped in several other crop species (Ori et al., 1997; Spielmeyer et al., 1998; Tekeoglu et al., 2000).

The objective of this study was to detect and map loci associated with resistance to FOP race 4 in a population of common bean RILs derived from an inter-racial cross between Durango and Mesoamerica, Belneb RR-1 x A55.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
A population of F6-derived RILs was developed from the common bean cross Belneb RR-1 x A55 through single seed descent (Ariyarathne et al., 1999). Common bean is a self-pollinating, diploid species with a haploid chromosome number of 11. This population was chosen because the parents have contrasting responses to FOP inoculation, and a linkage map was already available from a recent study that identified QTLs for halo blight (caused by Pseudomonas syringae pv. phaseolicola), common bacterial blight [caused by Xanthomonas campestris pv. phaseoli (Smith) Dye], and bean common mosaic virus resistance (Ariyarathne et al., 1999). The germplasm line Belneb RR-1 is a Great Northern bean derived mostly from germplasm representative of Durango (Stavely, 1989), and is highly susceptible to FOP race 4. A55 is representative of Mesoamerican germplasm, has small black seeds, and is resistant to FOP races 1, 2, and 4 (Silbernagel and Mills, 1991; Velazquez-Valle et al., 1997). Seventy-eight RILs at the F9 stage were used to generate the linkage map (Ariyarathne et al., 1999). Because of inadequate seed supply of two lines, only 76 RILs were analyzed for Fusarium wilt disease severity in the F10 generation.

Disease Evaluation
The inoculation technique was adapted from the root-dip procedure described by Pastor-Corrales and Abawi (1987) using FOP race 4 isolate B13 (ATCC 90245) (Salgado et al., 1995). B13 was isolated from infected bean tissue, and a single spore culture was maintained in a sterile sandy soil with 2% (w/v) powdered oats at 4°C until use. For inoculum preparation, the isolate was transferred to half-strength potato dextrose agar plates and grown at 22°C with a 12-h dark cycle for 14 to 21 d. Immediately before inoculation, plates were flooded with distilled water, scraped with a spatula, and the conidia suspended in distilled water. After vigorous mixing, the conidial suspension was filtered through two layers of cheesecloth to remove mycelium. Conidial concentration was adjusted to 106 per mL in distilled water using a haemocytometer.

Inoculations were performed in a greenhouse maintained at {approx}16/32°C night/day temperature with supplemental light to provide a 14-h daylength and 400 to 600 µmol m-2 s-2 of photosynthetically active radiation. Seeds were sown in Metro-mix 200 (Scott's, Marysville, OH) in 15-cm pots at a rate of six seeds per pot. Plants were inoculated {approx}2 wk after planting, when unifoliolate leaves were fully expanded. Plants were gently uprooted and the media removed from the roots by washing under tap water. Roots were then submerged in tap water for 5 min. Approximately one-third of the distal root mass was clipped with scissors, and the remaining roots submerged in the conidial suspension with mixing for 5 min. Plants were then transplanted into pre-moistened soil mix with 2 plants per 15-cm pot and watered lightly. Control plants were clipped, submerged in tap water for 5 min, then transplanted in the same manner. To reduce shock after inoculation, plants were placed under a shade cloth for 2 d and periodically misted. Plants were fertilized with 100 mL of a 0.3% solution of Miracle-gro (15-30-15, Scott's, Port Washington, NY) 1 and 2 wk postinoculation.

Fusarium wilt severity was scored 3 wk after inoculation, using a disease severity index (DSI) similar to one developed at the Centro Internacional de Agricultura Tropical (CIAT) (Pastor-Corrales and Abawi, 1987). Visual evaluation of foliar symptoms and internal examination of vascular tissues were used to assign a numerical DSI score as follows: 1 = no foliar or vascular symptoms, 3 = 1 to 10% of foliage symptomatic, mild plant stunting, and vascular discoloration in hypocotyls, 5 = 11 to 25% of foliage symptomatic, moderate plant stunting, and vascular discoloration extending to first stem node, 7 = 26 to 50% of foliage symptomatic, severe plant stunting, and vascular discoloration throughout stem and petiole, 9 = plant death.

For each RIL, we conducted two separate evaluations for reaction to FOP, each consisting of 10 inoculated plants and four noninoculated controls. Each evaluation was considered a replicate and the experiment was analyzed as a completely randomized design. Because of greenhouse space constraints, the RILs were evaluated in sets that included 13 to 28 RILs plus both parents as resistant and susceptible checks. Mean DSI scores for each RIL were calculated for each replicate, then replicate means were averaged to give an overall mean DSI score for the line. Lines with high standard deviations (>2) for DSI, within or between replicates, were reevaluated. The reevaluations were used as additional replicates in the final analysis. Lines were considered resistant with mean ratings of 1 to 3, intermediate with 3.1 to 6.9, and susceptible with 7 to 9 (Cross et al., 2000).

DNA Markers and Linkage Map Construction
DNA extraction, marker screening, and linkage map construction were conducted previously in a separate study at the University of Nebraska, Lincoln, Department of Horticulture, as described by Ariyarathne et al. (1999). In brief, DNA was extracted from fresh trifoliolate leaves of parents and RILs using the method of Skroch and Nienhuis (1995). A linkage map was constructed using MapMaker version 2.0 (Lander et al., 1987). The resulting map included 87 RAPD and 3 SCAR markers grouped into 11 LGs with a span of 755 cM. Segregation distortion was observed in 10% of the markers, including 6 out of 10 markers on LG 10, which had an excess of Belneb RR-1 alleles (Ariyarathne et al., 1999). The LGs defined in this population were aligned with those of two previously constructed RFLP maps (Freyre et al., 1998; Vallejos et al., 1999).

Statistical Methods
Analysis of variance using the SAS GLM procedure (SAS Institute, 1989) was performed on the phenotypic data, with RILs considered a random effect. Associations between individual marker loci and disease severity were tested with single-factor analysis of variance using the SAS GLM procedure, with a threshold significance level of P = 0.01 for tentative detection of a QTL. The R2 value (x100) from this analysis was interpreted as the percent phenotypic variation explained by the locus. Least squares means of genotypic classes were calculated with the LSMEANS option of the GLM procedure. We chose the most significant locus from each genomic region having significant marker–trait associations, and combined these loci in stepwise fashion in multiple-locus models (GLM procedure). Epistatic interactions between detected QTLs and all other marker loci were analyzed using the GLM procedure.

QTL Cartographer version 1.14 (Basten et al., 2000) was used for CIM to confirm the results of single-factor analysis of variance and to determine the most precise estimate of QTL number and position. Using Model 6 of the Zmapqtl option, we selected 10 background parameters based on forward-backward regression analysis, and specified a 1-cM walking speed and a 5-cM window. One thousand permutations of the data set were performed to determine the empirical threshold significance level for QTL detection at a genome-wise Type I error rate of 0.05 (Churchill and Doerge, 1994). This procedure for establishing the significance level takes into account specific characteristics of a data set, including genetic map density, segregation distortion, and the number and magnitude of segregating QTLs. For each significant QTL, we determined a 2-log of the odds (LOD) support interval, defined as the linkage map distance corresponding to a decrease in LOD score of 2.0 on either side of the peak of the LOD curve.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The distribution of Fusarium wilt disease severity scores in the common bean population Belneb RR-1 x A55 ranged from 1.9 to 9.0, with an average DSI score of 7.4. Of 76 RILs, 53 lines were susceptible, 16 intermediate, and 7 resistant (Fig. 1). Regardless of how the intermediate lines were classified, this distribution did not fit the ratio of 1 resistant:1 susceptible (P <0.003) expected for single gene inheritance in a RIL population. On the other hand, the distribution did not approach normality, as would be expected in classic polygenic inheritance. Parental DSI scores were consistent in each successive inoculation, with Belneb RR-1 averaging 9.0 ± 0.1 and A55 averaging 2.9 ± 0.2.



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  		Fig. 1. Frequency distribution of mean Fusarium wilt disease severity index (DSI) scores of common bean Belneb RR-1 x A55 RILs. Number of lines falling between DSI points are indicated. Mean parental DSI scores and standard error values are indicated with arrows.

 
Three loci were significantly associated with DSI score (P < 0.01) using single-factor analysis of variance. The most significant marker was U20.750 on LG 10, which accounted for 63.5% of the phenotypic variance for DSI score. Markers K10.700 on LG 11 and AD4.450 on LG 3 had R2 values of 10.8 and 10.1%, respectively. When the latter two loci were combined with U20.750 in multiple-locus models, only U20.750 retained significance at P < 0.05. Composite interval mapping eliminated markers K10.700 and AD4.450 from consideration because the permutation test set the empirical log-likelihood (LOD) significance threshold relatively high, at 8.16. This method detected a QTL directly at marker U20.750 with a LOD score of 23.9, and scores decreased substantially 1 cM to either side of this locus and at flanking markers (Fig. 2). The 2-LOD support interval for this QTL extends from position 4.0 to position 7.6 on LG 10. These results indicate tight linkage, perhaps as close as 1 cM, between a major QTL for Fusarium wilt resistance and marker U20.750. However, this inference should be interpreted cautiously, because the small population size (n = 76) may have resulted in reduced precision in QTL location and an upward bias in QTL effect estimation (Melchinger et al., 1998).



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  		Fig. 2. Log-likelihood ratio for QTL detection along common bean linkage group (LG) 10 showing the peak log of the odds (LOD) score at marker U20.750. Marker names are listed to the left of the LG diagram and cumulative cM distances are indicated to the right. Markers showing significant segregation distortion are indicated by asterisks (*, P < 0.05; **, P < 0.01).

 
Although disease resistance loci frequently occur in clusters in common bean and other crops (Grube et al., 2000; Geffroy et al., 1999), the U20.750 locus does not occur in close proximity to known groupings of resistance loci or mapped nucleotide binding site-leucine-rich repeat (NBS-LRR) motifs (Rivkin et al., 1998). The closest resistance locus is a QTL for halo blight (HB 16) located {approx}7 cM away on LG 10 (Ariyarathne et al., 1999).

The average DSI score for RILs with the A55 marker genotype at the U20.750 locus was 3.6 points lower than those with the Belneb RR-1 genotype. Although A55 was determined to be the source of the resistant allele, we could not determine if the allele was dominant or recessive because of the homozygous nature of the RILs and the lack of F1 plants in our screening. Examination of the DSI score distribution by genotype class at U20.750 revealed an unexpected pattern (Fig. 3). Virtually all lines with the Belneb RR-1 allele at this locus were in the susceptible range as expected, but individuals with the A55 allele spanned the range from resistant to susceptible classes.



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  		Fig. 3. Frequency of A55 and Belneb RR-1 alleles at locus U20.750 for common bean Fusarium wilt disease severity classes.

 
There are several possible explanations for this pattern of expression of resistance, the most likely being that the A55 allele is necessary, but not sufficient for resistance. Similar results showing partial resistance conferred by a major locus have been documented in QTL mapping of anthracnose resistance in common bean (Geffroy et al., 2000) and in Fusarium wilt resistance in tomato (Lycopersicon esculentum Mill.) (Ori et al., 1997). It is possible that additional QTLs are present in our mapping population, as initially indicated by single-factor analysis, but were not detected in subsequent, more stringent analyses. Other QTLs may be located in regions not covered by markers in the linkage map, such as the gaps present on LG 9 and LG 4 (Ariyarathne et al., 1999). There may also be epistatic interactions involving the major QTL on LG 10 that affected the frequency distribution. Although this population is small for the analysis of epistasis, we did detect significant (P = 0.0001) interaction between U20.750 and locus AN15.400 on LG 1. Interestingly, it is the susceptible parent allele at marker AN15.400 that lends an additional 1.5 DSI points towards resistance in this interaction. This result is similar to several other QTL studies, in which beneficial alleles were contributed by the presumed inferior parent (Bernacchi et al., 1998; Byrne et al., 1996). Additional epistatic loci were detected on LG 2, LG 6, and LG 9 (0.001 < P < 0.01). Other possible explanations for the phenotypic distribution of lines with the A55 allele are (i) recombination between the marker and the QTL, although this seems unlikely due to the close linkage revealed by CIM; (ii) errors in marker scoring; and (iii) DSI score variability observed within some RILs.

Although previous reports have indicated that Fusarium wilt resistance is qualitative in Durango and quantitative in Mesoamerica (Salgado et al., 1995; Cross et al., 2000), our study found a resistance locus from the Mesoamerican parent (A55) acting in a primarily qualitative manner. However, our results also suggested that additional genes or genetic interactions may be involved in resistance in this population, a finding that agrees with previous reports of quantitative inheritance. Because the earlier studies detected the effects of, but did not localize, Fusarium wilt resistance loci, we cannot state whether the LG 10 locus identified in this study corresponds to the previously reported loci. Given the diversity of germplasm within the Mesoamerican race (Singh et al., 1991), it is not surprising that a variety of resistance genes and mechanisms might exist.

In conclusion, a major locus for Fusarium wilt resistance has been identified and mapped for the first time in a common bean population. Marker U20.750 is a good candidate for conversion to a more reliable SCAR marker for use in marker-assisted selection to eliminate the majority of Fusarium wilt susceptible genotypes (Kelly and Miklas, 1999). For example, in the present study, 88% of the susceptible RILs, or 68% of all RILs, might have been eliminated through DNA genotyping, significantly reducing the amount of disease screening required. In future studies, this population will be screened with different races of FOP to determine race-specificity of this locus, and inoculation in the field will be used to confirm the involvement of the U20.750 locus in field-level resistance.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge financial support from the Colorado Institute for Research in Biotechnology and the Colorado Agricultural Experiment Station. This research was conducted in collaboration with the W-150 Regional Project on genetic improvement of beans for disease resistance and food value.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Published as Paper no. 13212 Journal Series, Nebraska Agricultural Division, Univ. of Nebraska, Lincoln, NE.

Received for publication November 9, 2000.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 


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