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a Rhinelander Agricultural Research Station, Univ. of Wisconsin, Rhinelander, WI 54501
b Monsanto Co., St. Louis, MO 63167
c Dep. of Plant, Soil, and Insect Sciences, Univ. of Massachusetts, Amherst, MA 01003
d Dep. of Horticulture, Univ. of Wisconsin, 1575 Linden Dr., Madison, WI 53706
* Corresponding author (nienhuis{at}wisc.edu).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: BBS, bacterial brown spot • IBC, inbred backcross • Pss, Pseudomonas syringae pv. syringae • QTL, quantitative trait loci • RAPD, random amplified polymorphic DNA • RIL, recombinant inbred line
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Seed transmission of BBS is possible (Thaung and Walker, 1957); however, its efficiency as a significant source of primary inoculum to start an epidemic is low (Hagedorn and Inglis, 1986). Soil transmission due to overwintering of Pss in Wisconsin soils is also low (Hoitink et al., 1968). The most important mode of dispersal of Pss is the wind (Lindemann et al., 1984; Lindemann and Upper, 1985; Upper and Hirano, 2002); however, rain and overhead irrigation have also been identified as effective dispersal mechanisms of Pss (Constantinidou et al., 1990; Hirano and Upper, 1995). Intense rainfall events have been found to trigger the growth of Pss populations on bean leaves (Hirano et al., 1996). Highly virulent epiphytic populations of Pss have been observed to overwinter on leaves of hairy vetch (Vicia villosa Roth), a common leguminous weed in snap bean production areas of central Wisconsin (Ercolani et al., 1974), providing an important source of inoculum that often results in outbreaks of BBS in adjacent bean fields.
Recommendations for controlling BBS include crop rotation, the use of resistant cultivars, control of weed hosts, especially hairy vetch, and the use of certified seed from arid seed production areas (Hagedorn and Inglis, 1986; Pike et al., 2003). Chemical seed treatments and foliar sprays have provided erratic control of the disease (Pike et al., 2003). The development of resistant cultivars has been identified as a priority for the U.S. North Central region (Pike et al., 2003).
Sources of resistance to Pss have been identified in Phaseolus vulgaris among plant introductions collected in Mexico (Hagedorn and Rand, 1977; Antonius and Hagedorn, 1979). Backcross breeding using these resistant sources as the donor parent resulted in the development of a series of resistant parental lines including Wisc. BBSR-130, BBSR-133, BBSR-17, and BBSR-28 (Hagedorn and Rand, 1977, 1980; Antonius and Hagedorn, 1979; Daub and Hagedorn, 1979). More recently, Puebla 152, a Mexican black bean variety, was identified as a source of resistance (Kmiecik, 1991).
Variability for BBS resistance can be evaluated by counting the number of lesions on a random sample of leaves harvested from the canopy. The ability of leaf surfaces to sustain epiphytic populations of Pss can be estimated by dilution plating of epiphytic Pss populations washed off the leaf surface (Crosse, 1959; Leben, 1965). Differences among Pss populations can be used to distinguish resistant from susceptible snap bean lines in the presence or absence of leaf lesions (Daub and Hagedorn, 1981); however, this technique is very laborious. An indirect method of measuring Pss populations on leaf surfaces was developed by Hirano et al. (1985), based on the discovery in the early 1970s that Pss was responsible for ice nucleation of super-cooled water (Maki et al., 1974; Lindow, 1983; Upper and Vali, 1995). The test tube ice nucleation method can be used as a rapid means of estimating populations of epiphytic ice nucleation active bacteria and can estimate the probability of appearance of BBS symptoms within 4 to 8 d (Hirano et al., 1987). The test tube ice nucleation technique has proven suitable to discriminate among germplasm with different susceptibility levels (Kmiecik et al., 1990; Hirano et al., 1996).
Information on BBS epidemiology is available from studies of the interaction of Pss with susceptible snap bean hosts, providing a better understanding of the role of Pss pathogenesis genes on snap bean (Hirano and Upper, 2002). In contrast, very little has been published with regard to the inheritance and possible mechanisms of host resistance (Daub and Hagedorn, 1979; Hagedorn and Rand, 1980; Jung et al., 2003). The most relevant inheritance study of BBS resistance using a segregating population was done by Jung et al. (2003). This research used the Hirano et al. (1987) technique on field-grown plants and the Pss seedling stem inoculation method in a Belneb RR-1 x A55 RIL dry bean population and concluded that genomic regions located in several linkage groups were associated with BBS resistance.
In the present study, test tube ice nucleation and field evaluations were used to identify and confirm QTL associated with BBS resistance using RIL and IBC bean populations that share Puebla 152 as the common donor parent.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Test Tube Ice Nucleation Assay
Sixty-two BC1F4 families corresponding to the EEP-IBC population were planted at the Arlington Agricultural Research Station in the summer of 1996 in a randomized complete block design with four replications. Plots were overplanted and thinned to 16 plants spaced approximately 0.15 m apart. Rows were approximately 0.75 m apart. No supplemental irrigation was applied.
Sixteen leaflets were randomly sampled from the top of the canopy of each plot from 0600 to 0800 h, placed in coolers containing bags of ice, and immediately processed using the test tube ice nucleation assay. The number of tubes in each plot in which an ice nucleation event (frost) occurred at –2.5°C was recorded (Hirano et al., 1985). The number of BBS lesions on each sample of 16 leaflets from each plot was counted.
In 2000 and 2001, 16 leaflets from 70 EP-RIL families and the parents (Eagle and Puebla 152), were evaluated for ice nucleation temperature using a modification of the method described in Hirano et al. (1985, 1987). Individual leaflets were put into 16-mm test tubes containing 9 mL of a sterile potassium phosphate buffer (0.01 M, pH 7.0) and randomly assigned to one of the 192 possible positions in six racks immersed in an ethanol bath. While in the racks, each test tube position was held by a metal support padded with silicone rubber forming a well that contained thermocouples used to scan the temperature of each test tube. The cooling rate in the ethanol bath was 
0.04°C and was maintained manually by controlling the flux of alcohol pumped from a flow-through cooling system. The temperature of each tube was monitored every 12 s with a CR7 data logger (Campbell Scientific, Logan, UT) and transmitted to a computer equipped with software that recognized and recorded ice nucleation events as a sharp rise in the temperature associated with the release of thermal energy by ice nuclei. The number of BBS lesions was based on a sample of 16 leaflets from each plot immediately after test tube ice nucleation assays were performed.
Experimental Designs and Field Evaluation
The EP-RIL population was field evaluated at the Arlington Agricultural Research Station for 3 yr. In 1996, the EP-RIL population experiment consisted of three replications of 64 lines using a randomized complete block design. In 2001, 70 EP-RIL were planted along with the respective Eagle and Puebla 152 parents as checks using a blocks within replication design (Schutz and Cockerham, 1966) that included six blocks of 12 lines. In 2002, both the EP-RIL and the EEP-IBC populations were field evaluated for lesion counts on the middle leaf of trifoliates sampled from the canopy. The EP-RIL experiment consisted of three replicates including the parental checks.
Seventy-two entries, including the parental checks, were evaluated in the 2002 EEP-IBC population using a blocks within replication design with three replications. The experimental plots consisted of one row of 16 plants. Every fourth row was planted with the cultivar Eagle used as the disease spreader. The Eagle spreader rows were planted 1 wk before planting the experimental plots. Infection of the Eagle spreader rows was enhanced by immersing seeds for 1 min before planting in a wetable powder containing 500 mg of bean leaf powder collected from BBS-infested leaves in a previous season and 200 g of laboratory-grade talcum powder (Fisher Scientific, Fairlawn, NJ) added to 1 L of water for 1 kg of seed (S.S. Hirano, Dep. of Plant Pathology, Univ. of Wisconsin, Madison, personal communication, 2002). The BBS lesion counts were taken once, in the same day, between the bloom and pod set stages, when visual differences in the number of BBS lesions among lines were apparent.
Statistical Analysis of Field Data
Analyses of variance and QTL analyses were performed on the EP-RIL population using the ice nucleation temperature means per plot for 2000 and 2001 and normalized values for the number of BBS lesions, using the Box–Cox family of transformations (Box and Cox, 1964). The Arc 1.04 software (Cook and Weisberg, 2002) was used to find the power transformations that allow variables to approach normality. Analyses of variance using transformed field BBS data were computed using SAS PROC MIXED procedures (SAS Institute, Cary, NC). Predicted means (best linear unbiased predictors) were obtained for each random EP-RIL family used for QTL search. Spearman rank correlations were used to estimate the product-moment correlations among ice nucleation temperature and field data corresponding to the same or different years without any assumption regarding the distribution of the data or homogeneity of variances.
Variance Components, Heritability Estimates, and Confidence Intervals
Variance components for the analyses of variance were computed from the mean square errors of the analyses of variance for the transformed number of BBS lesions or ice nucleation temperature in the EP-RIL (F2:8) and EEP-IBC (BC1F7) populations according to the expectations for random models (Cockerham, 1963; Knapp et al., 1985). Narrow-sense heritability estimates were made on a progeny-mean basis using the procedures of Hallauer and Miranda (1988); confidence intervals of heritability were calculated with an F test proposed by Knapp et al. (1985). Heritability estimates computed from inbred lines are considered narrow-sense estimates because it can be assumed that the dominance variance and the covariance of additive and dominance effects are both equal to zero in the EP-RIL F6:8 and inbred backcross BC1F7 lines (Cockerham, 1983). Under these assumptions, the genetic variance for the lines can be translated to covariance of relatives that is equivalent to additive variance and additive x additive interactions. Estimates of narrow-sense heritability may be overestimated due to epistatic relationships among additive effects (Cockerham, 1983).
Identification of Quantitative Trait Loci in the Eagle x Puebla 152 Recombinant Inbred Line Population
A molecular map consisting of 357 RAPD markers spanning 764.6 cM (Skroch, 1998) was used to identify marker–QTL association for ice nucleation active bacterial populations and the transformed number of BBS lesions on the EP-RIL population. Composite interval mapping (Zeng, 1994) with a forward selection–backward elimination multiple regression approach was performed using 70 EP-RIL and the transformed predicted means for tube nucleation temperature and number of BBS lesions obtained in each experiment. The focus of the QTL identification was to estimate QTL and additive effects. The window size was set to 10 cM and the number of markers for background control was five. Quantitative trait locus analyses were performed using Windows QTL Cartographer (Wang et al., 2004).
Confirmation of Quantitative Trait Loci for Bacterial Brown Spot Resistance
To confirm the QTL associations identified in EP-RIL population, the EEP-IBC population was screened using 26 10-mer RAPD primers (Operon Biotechnologies, Huntsville, AL).
The DNA was extracted from a random sample of six plants from each family in the EEP-IBC population using a DNA extraction procedure developed by Jhingan (1992) and modified by Johns et al. (1997). Polymerase chain reactions (PCR) were performed in 12-µL volumes (6 µL of PCR mix + 6 µL of standardized DNA solution), in an MJ Research PTC 100 thermocycler (Bio-Rad Laboratories, Hercules, CA), following procedures used by Johns et al. (1997), and the PCR products were visualized in 1.5% agarose gels.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Significant Spearman rank correlations were also observed for the comparison of BBS field data and leaf ice nucleation temperature ranks. The higher correlations were observed between the 1996 ice nucleation temperature and 1996, 2001, and 2002 field data, with rs = 0.65, 0.60, and 0.58, respectively, and P < 0.0001 (Table 2). These results confirm that ice nucleation temperature can consistently discriminate between resistant and susceptible lines.
Significant variation for the number of BBS lesions on the middle triafoliate leaves was also observed in the EEP-IBC population in 2002 (Table 1). The heritability estimate for the number of BBS lesions on leaves was 0.78 for the EEP-IBC population. This estimate was in close agreement with the heritability estimate obtained in the EP-RIL for the same year (h2 = 0.85).
Mapping Quantitative Trait Loci for Bacterial Brown Spot Resistance in the Recombinant Inbred Line Population
A RAPD map for the EP-RIL population developed by Skroch (1998) was used to investigate QTL associated with variation for ice nucleation temperature and predicted number of BBS lesions. Marker–QTL relationships were considered potentially useful as indirect selection criteria when significant associations were consistently detected across years for ice nucleation temperature, number of BBS lesions, or both traits (Table 3).
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The correspondence of ice nucleation temperatures with the number of BBS lesions confirms that ice nucleation temperature is a valid surrogate variable to estimate variability on Pss populations (Daub and Hagedorn, 1981; Hirano et al., 1996).
Confirmation of Quantitative Trait Loci for Bacterial Brown Spot Resistance in the Inbred Backcross Population
A BC1F7 inbred backcross population (EEP-IBC) derived from the same parents as the EP-RIL population was used as a related independent population to confirm QTL associations on linkage groups B1, B3, B6, and B11 identified in the EP-RIL population.
A QTL from linkage group B1, near RAPD marker P1.1500, that explained 13% of the variability for ice nucleation temperature in 2001 in the EP-RIL population was also significant in the EEP-IBC population, explaining 8% of the variation for the number of BBS lesions in 2002.
A QTL for resistance located in linkage group B3 close to RAPD markers V10.450, O10.350, and F10.900 was not confirmed by the ice nucleation and field data obtained in the EEP-IBC population in 1996 and 2002, respectively. Inbreeding of the EEP BC1F1 backcross was expected to result in approximately 28.1 and 25.8% of the EEP BC1F4 and BC1F7 lines showing the presence of each marker. Only four lines in the EEP BC1F4 and BC1F7 generations resulted in the presence of these markers in both years. This disequilibrium may have been due to unintentional selection against this region in linkage group B3 during the development of the EEP-IBC population (Fig. 2b, Table 3); however, Jung et al. (2003) suggested the importance of markers AD4.100 and R20.400 for ice nucleation temperature and number of BBS lesions in the field using a RIL population derived from the cross of the common bean Belneb RR-1 (susceptible to BBS) x A55 (resistant to BBS). This region depicted by Jung et al. (2003) spanned the same region of linkage group B3 identified in this study. To confirm the importance of the association of markers V10.450, O10.350, and F10.900 with QTL for BBS resistance, introgression of chromosomal segments carrying this marker should be made to a larger number of families and the resulting progenies should be field tested for both the Pss populations and the number of BBS lesions. Jung et al. (2003) also suggested two additional regions located in linkage group B3, one identified by markers R2.1200 and K10.1050, and the other by R2.430 and AD4.450. The limitation to comparing these additional regions along with the findings of this study is that R2.1200, K10.1050, R2.430, and AD4.450 do not map to B3 in the Eagle x Puebla and core map. In addition, no mapping distances among these markers were given by Jung et al. (2003).
A chromosomal region in linkage group B6 near RAPD marker O10.650 was significantly associated with a reduced number of BBS lesions and a reduced ice nucleation temperature in the EP-RIL population in 1996. The same marker was associated with a reduced number of BBS lesions for EP-RIL in 2002. The importance of this chromosomal region as a source of variation was confirmed by its association with an increased number of BBS lesions in the EEP-IBC population. In addition, significant association of the AK6.1500 RAPD marker with a decreased number of BBS lesions was observed in EEP-IBC in 2002 (Table 3). Results suggest that RAPD marker AK6.1500 is also associated with reduced ice nucleation temperature and that marker AN6.1600 is associated with a reduced ice nucleation and reduced number of BBS lesions. Markers AK6.1500 and AN6.1600 are both derived from Puebla 152 and mapped to the same region, 4.1 cM away from marker O10.650 (Skroch, 1998). Jung et al. (2003) reported that O10.650 was also significantly associated with variation in Pss through stem inoculation of a RIL population derived from the cross of the common bean lines Belneb RR-1 x A55. Marker O10.650 is derived from the susceptible Eagle parent. As discussed above, the association between O10.650 and a reduced number of BBS lesions is probably due to linkage disequilibrium between O10.650 and Puebla 152 inherited segments represented by AN6.1600 and AK6.1500 (Table 3).
A QTL for BBS susceptibility was identified in a region located in linkage group B11, close to RAPD markers H20.950 and AL15.1300. This region was associated with increased ice nucleation temperature and number of BBS lesions in the 1996 and 2001 analyses of the EP-RIL population; however, this was not confirmed in the EEP-IBC population. In the EEP-IBC population, this region resulted in a weak, nonsignificant association for increased ice nucleation in the EEP BC1F4 population evaluated in 1996 (R2 = 0.04, P = 0.0785).
In this study, two regions of the bean genome have been identified and confirmed to carry QTL associated with resistance to BBS based on the number of BBS lesions and ice nucleation temperature. A QTL on linkage group B1, flanked by RAPD marker P1.1500, resulted in significant associations in the EP-RIL and EEP-IBC populations. Significant associations confirmed the importance of a region of linkage group B6, near the linked markers AN6.1600 and AK6.1500, explaining 35% of the variability observed for the number of BBS lesions in the EEP-IBC population in 2002, confirming results from a previous study (Jung et al., 2003). A QTL identified in linkage group B11 was not confirmed in the EEP-IBC population because too few lines carried the necessary markers near the QTL in this population (Fig. 2c).
Studies performed primarily at the University of Wisconsin-Madison and University of California-Berkeley have generated important information on the relationship between snap bean and Pseudomonas syringae pv. syringae (Hirano and Upper, 2000), genetics of its pathogenicity (Hirano et al., 1997, 2001), and proposed models for leaf colonization and pathogenicity (Atkinson and Baker, 1987a, 1987b). It is likely that, due to the nature of the Pss–snap bean relationship, genes located in several Phaseolus vulgaris chromosomal regions affect the fitness of the bacterium, limiting its epyphitic or pathogenic characteristics, or both. The present work may lead to new hypotheses by examining the host–pathogen interaction in the context of an array of genetic variability that can be generated in the host with the introgression of QTL for resistance.
Due to variation in disease incidence across years, locations, and seasons, gain from selection based on field evaluation of BBS disease resistance can be sporadic and inefficient. The test tube ice nucleation assay is an indirect selection criterion highly correlated with field symptomalogy; nevertheless, it is very time and labor intensive and may be impractical for evaluation of large populations in a breeding program. Selection based on RAPD markers P1.1500 and AN6.1600, or AK6.1500 associated with QTL for BBS resistance can be more reliable than field evaluation and more efficient than the test tube ice nucleation assay. The RAPD markers identified in this study are reproducible and robust in their identification across populations and laboratories and RAPD technology is a relatively inexpensive and accessible molecular technique. Random amplified polymorphic DNA markers can be used efficiently in the winter season to screen individual seedlings in segregating generations to identify those individuals that possess the desired RAPD banding pattern. The seedlings of selected plants can be grown to maturity in a greenhouse and sufficient seed harvested for confirmation of resistance the following summer in the field using replicated trials of selfed progeny. The field trials include a manageable number of entries (often <100 from a population of >1000), all of which have an increased probability of possessing RAPD-based QTL associated with BBS resistance. Field evaluation and confirmation among a manageable number of progeny is especially important in snap bean breeding programs that use exotic donor parents, such as Puebla 152, because it permits tandem selection for disease resistance and characteristics associated with processing vegetable quality.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication May 3, 2006.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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