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a USDA-ARS, Vegetable and Forage Crops Research Unit, 24106 N. Bunn Rd., Prosser, WA 99350
b Dep. of Crop and Soil Sciences, Michigan State Univ., East Lansing, MI 48824
c Pioneer Hi-Bred International, Inc., 7250 NW 62nd Ave, Johnston, IA 50131
d Dep. of Plant Sciences, North Dakota State Univ., Fargo, ND 58105
* Corresponding author (pmiklas{at}pars.ars.usda.gov)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: QTL, quantitative trait loci • RIL, recombinant inbred line • SRAP, sequence related amplified polymorphism • sCIM, simple composite interval mapping • MAS, marker-assisted selection
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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An integrated strategy encompassing crop rotation, minimum fertilizer rate, reduced irrigation, timely fungicide application, and less susceptible cultivar, is used to manage white mold disease in bean (Schwartz and Steadman, 1989). A few pinto cultivars, Chase (Coyne et al., 1994) and Maverick (Grafton et al., 1997), possess a low level of partial resistance to white mold. Pinto bean cultivars with moderate to high levels of partial resistance have not been developed yet because resistance is difficult to breed for, due in part to low heritability (Fuller et al., 1984; Lyons et al., 1987; Kolkman and Kelly, 2002; Miklas and Grafton, 1992; Miklas et al., 2004; Park et al., 2001). The lack of adapted resistance sources also slows progress in breeding for improved resistance.
Moreover, breeding for genetic resistance is complex because it is conditioned by both avoidance and physiological mechanisms (Miklas et al., 2001). The use of avoidance mechanisms, including upright and open plant structure, less dense canopies and branching patterns, elevated pod set, and reduced lodging have been shown to reduce white mold damage (Schwartz et al., 1987; Kolkman and Kelly, 2002). These avoidance traits enhance penetration of the canopy by sun and aid air circulation, thereby creating a microclimate that is less conducive for infection and disease progression. Generally, most resistance detected by a laboratory or greenhouse screening method has a physiological basis. The stay green stem trait (Miklas et al., 2004), phytoalexin response (Sutton and Deverall, 1984; Miklas et al., 1993), and insensitivity to oxalic acid (Kolkman and Kelly, 2000; Chipps et al., 2005) represent physiological mechanisms implicated in genetic resistance to white mold disease.
A recent goal of many bean breeding programs has been to identify, tag, and map QTL for resistance to white mold to better understand the genetics underpinning partial resistance and to facilitate marker-assisted breeding for partial resistance obtained from different sources: navy ‘ICA Bunsi’, landrace G122 (PI 163120) from India, snap bean breeding line NY6020–4, and pompadour cultivar PC 50. Miklas et al. (2001) identified a QTL derived from G122 on linkage group B7 conditioning physiological resistance and on B1 contributing to disease avoidance. Similarly, two QTL from NY6020–4 were found on B6 associated with disease avoidance and on B8 conditioning physiological resistance (Miklas et al., 2003). Three major-effect QTL from PC-50 expressed in the greenhouse straw test and field were mapped to linkage groups B4, B7, and B8 (Park et al., 2001). Some of the resistance genes may be in common, as the B7 and B8 QTL from PC-50 map to the same general location as the B7 QTL from G122 and the B8 QTL from NY6020–4 (Miklas et al., 2006c). A preliminary report (Miklas, 2006) showed that marker-assisted backcrossing was successful in transferring white mold resistance conferred by B7 QTL from G122 and B8 QTL from NY6020–4 into susceptible pinto bean.
ICA Bunsi has been used extensively in breeding for white mold resistance in navy bean. The ICA Bunsi source of resistance segregating in crosses with susceptible navy ‘Newport’ was conditioned by QTL on linkage groups B2 and B7, and with susceptible black ‘Raven’ by QTL on B2, B5, B7 and B8 (Kolkman and Kelly, 2003; Ender and Kelly, 2005). The major-effect QTL residing on B2 and B7 were expressed in both populations. The QTL on B2 and B7 were confirmed by Kolkman and Kelly (2003) in a second navy bean population ‘Huron’/Newport. The B2 QTL was associated with partial resistance expressed in the field; whereas, the B7 QTL was associated with resistance to oxalate in a greenhouse test (Kolkman and Kelly, 2000). The B7 QTL, independent of the QTL identified near the Phs locus by Miklas et al. (2001), was also significantly associated with agronomic traits including yield, seed size, lodging, and days to flower (Kolkman and Kelly, 2003).
To investigate the potential for ICA Bunsi as a source of white mold resistance in pinto bean improvement, Miklas et al. (2004) examined the inheritance of ICA Bunsi-derived resistance in a navy x pinto bean recombinant inbred line population. They observed that partial field resistance had moderate heritability, was influenced by the environment, and was likely conditioned by genes with small effects. The stay-green trait where pods reach maturity but the plant remains green and physiologically active was associated with partial resistance in the population. A recombinant inbred line from the population, ‘AN-37’, which possessed pinto seed type and a high level of partial resistance to white mold was released as a germplasm line USPT-WM-1 (Miklas et al., 2006a). The objectives of this study were: i) to identify QTL conditioning partial resistance in the ICA-Bunsi derived navy x pinto population developed by Miklas et al. (2004), and ii) to examine resistance-linked QTL for association with avoidance and agronomic traits.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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The 85 F5:8 RILs were previously evaluated by Miklas et al. (2004) for reaction to white mold in four field environments: Hatton, ND, in 2001; Carrington, ND, in 2002; and in Paterson, WA, in 2001 and 2002. The planting dates, experimental design, plot size, planting density, weed control, fertilizer for optimum plant growth, and irrigation to maintain optimum moisture conditions for white mold infection, were as previously described.
A brief description of the phenotypic traits measured in these trials by Miklas et al. (2004) is given below. Disease reaction was scored from 1 to 9 based on combined incidence and severity of infection at physiological maturity (defined as 80% of the pods at harvest maturity), where 1 = no diseased plants and 9 = 80–100% diseased plants and/or 60–100% infected tissue (Miklas et al., 2001). Disease avoidance traits measured included: canopy porosity (Deshpande, 1992) at mid-pod fill and scored from 1 to 5, where 1 = a porous canopy with the soil surface highly visible, and 5 = dense canopy with no soil visible; canopy height (cm) at mid-pod fill; and lodging scored at physiological maturity from 1 to 9; where 1 = no lodging and 9 = > 90% lodged. Lodging was not obtained for the WA 2002 trial. Maturity (d) was recorded as the number of days from planting to physiological maturity. Stay-green stem, only recorded for the ND trials, was scored from 1 to 5 at physiological maturity; where 1 = 0–20% green stem and 5 = 80–100% green stem. Plot yield (kg ha–1) and seed weight (g 100 seeds–1) were measured.
A composite leaf tissue sample was collected from the first emerging trifoliolate leaves of four plants of each line. Genomic DNA was extracted using the FastDNA Kit (Bio 101, Vista, CA) according to the manufacturer's instructions. The purified DNA was adjusted to 10 ng µL–1 using a fluorometer before all PCR reactions. Equal amounts of DNA from the seven most resistant and seven most susceptible RILs as determined by average disease reaction across all four field tests were used to form resistant (R-bulk) and susceptible (S-bulk) DNA bulk samples, respectively. Similarly to the multi-trait bulking strategy described by Kolkman and Kelly (2003), other traits were considered in the formation of the DNA bulk samples, as above average yield and average maturity were sought for individual RILs comprising the resistant bulk. The multi-trait bulk segregant analysis was used to target identification of QTL conditioning resistance in an acceptable phenotype, lacking such undesirable traits as low yield and late maturity. Lines with inherently low yield will escape white mold disease primarily as a result of less biomass enabling air and light penetration of the canopy. Later maturity was significantly correlated with less disease in the Aztec/ND88–106–04 RIL population (Miklas et al., 2004).
The R- and S-bulks were screened against 650 decamer primers (Operon Technologies, Alameda, CA) for presence of random amplified polymorphic DNA (RAPD) markers. PCR consisted of 25 µL-reactions containing 2 U Stoffel fragment DNA polymerase (Applied Biosystems, Foster City, CA), 1 X Stoffel buffer, 0.2 µM primer, 5 mM MgCl2, 200 µM each dNTP, and 25 ng template DNA. Amplifications were performed on a Peltier Thermal Cycler PTC-200 (MJ Research Inc., Waltham, MA) programmed for 2 min at 94°C; 3 cycles of 1min at 94°C, 1 min at 32°C, 1 min at 72°C; 30 cycles of 10 s at 94°C, 20 s at 37°C, 2 min at 72°C; 5 min at 72°C. RAPDs observed between the bulks were assayed across the individuals that comprised the bulks. RAPDs that cosegregated with disease reaction in at least 78% of the individuals comprising the DNA bulks were subsequently assayed across the entire population of 85 RILs.
Other phenotypic traits and markers that were mapped across the whole population included: the I gene for resistance to Bean common mosaic virus on linkage group B2 detected by segregation for top necrosis (ND88–106–04) vs. mosaic (Aztec) using strain NL-3 D and linked sequence characterized amplified region (SCAR) marker SW13.690; the P gene on B7 detected by segregation for white seed color (ND88–106–04) versus colored seed (Aztec); Asp gene on B7 conferring seed brilliance detected by shiny (ND88–106–04) vs. dull (Aztec) seed coat appearance; the SAP6.820 and BC409.1250 SCARs linked with a QTL for resistance to common bacterial blight [caused by Xanthomonas axonopodis pv. phaseoli (Smith) Vauterin et al.] on B10, although both parents lacked the resistance QTL the markers segregated anyway; and the Ur-3 gene for resistance to rust caused by Uromyces appendiculatus (Pers.) Unger var. appendiculatus on B11 detected by hypersensitive fleck (ND88–106–04) vs. large pustule reaction (Aztec) to Race 53 and linked SCAR SK14.620. RAPDs identified between a different pair of R- and S-DNA bulks from the Aztec/ND88–106–04 population designed for presence and absence of the Ur-3 gene as part of another study were also mapped across the whole population and included in this study. The annealing temperatures and cycling profiles for the SCARs are listed online at http://www.ars.usda.gov/SP2UserFiles/Place/53540000/miklas/SCARtable.pdf (verified 23 Oct. 2006).
Following DNA sequencer procedures of Miklas et al. (2006b), 18 target region amplified polymorphism (TRAP) markers (Hu and Vick, 2003) were mapped across the entire population. The fixed primers for the TRAP protocol were based on published disease-related gene sequence from a Helianthus EST (LRR domain) 5'-GAAAGACGAAGGAACAGG-3', and a bean resistance gene analog RGA1 (NBS domain) 5'-CCTAAATGGGAGGAAGTG-3'. The same arbitrary primer 5'-GGAACCAAACACATGAAGA-'3 from a sequence related amplified polymorphism (SRAP) primer list published by Li et al. (2003) was paired with each fixed primer.
Eleven AFLP markers that were linked with QTL conditioning resistance to white mold derived from ICA Bunsi in a cross with a susceptible black bean ‘Raven’ were assayed across the whole population of Aztec/ND88–106–04 RILs following the protocol of Ender and Kelly (2005). Only five of the 11 AFLPs from the Ender and Kelly (2005) study mapped in the Aztec/ND88–106–04 population, including EAGAMCAT.165 on linkage group B2 (named AFLP 1 in this study), EAGGMCAA.170 and EAGTMCAT.360 on B5, EACAMCCG.460 on B8, and EAGTMCTT.300 (named AFLP 10) on partial linkage group LG-5, were not integrated with the core map (Ender, 2003). AFLPs associated with the major QTL for resistance in the Bunsi/Raven population on B7 were monomorphic in the Aztec/ND88–106–04 population.
A partial linkage map of the mapped markers was constructed using Mapmaker 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 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 (cM) distances between linked loci were based on recombination fractions using the Kosambi (1944) mapping function. The mapping of QTL-linked RAPDs across the BAT 93/Jalo EEP 558 RIL population (BJ) was used to anchor partial linkage groups obtained in this study to the core P. vulgaris map (Freyre et al., 1998). Seventy-one of the RILs from the BJ population were available for this purpose.
Association of markers with disease score and other traits was determined by linear regression of the trait means on individual marker genotypes using PROC GLM (SAS Institute, 1987). An F-test significant at P 
0.01 for a trait measured at a single location was used to indicate linkage between a marker and QTL. If significant for one location, a QTL was considered significant at P 0.05 level for the other locations.
For the partial linkage groups, simple composite interval mapping (sCIM) performed by MQTL (Tinker and Mather, 1995) across location means, was also used to detect QTL with main effects for partial resistance in the field as measured by disease score: for canopy porosity, canopy height, lodging associated with disease avoidance, for stay green stem, harvest maturity, yield and seed weight. A significant QTL was declared if the test statistic calculated by MQTL was greater than the significance threshold determined by permutation analyses (1000 permutations) of the data sets for each trait based on a 5% experiment-wise error rate (Doerge and Rebai, 1996). The test statistic calculated by MQTL was multiplied by 0.22 to equate to a LOD score.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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In addition to proximity to fungal defense-related genes, partial resistance conferred by the B2 QTL likely has a physiological basis because it is not influenced by disease avoidance traits (open canopy, plant height). The association of this QTL with reduced lodging at Carrington was not consistent across locations (Table 1), and was not detected by composite interval mapping (Fig. 2). The ND88–106–04 allele contributed to both reduced disease severity and reduced lodging. This B2 QTL was also associated with lodging in other populations (Kolkman and Kelly, 2003; Ender and Kelly, 2005). In those populations reduced lodging was contributed by the white mold susceptible parents (Newport and Raven) with upright architecture; whereas, reduced disease severity was contributed by the white mold resistant parent ICA Bunsi with a more prostrate indeterminate plant growth habit. Furthermore, interval mapping in those populations depicted a distance of about 10 to 12 cM between the LOD peaks for lodging and disease severity traits; thus, as suggested by Kolkman and Kelly (2003), separate mechanisms contributing to less disease from each parent were located in the same general region of the linkage group.
Composite interval mapping showed a strong association between the B2 QTL and expression of the stay green stem trait, which further supports a physiological basis for the partial resistance conferred by this QTL. Plants with stay green trait, described as pods reaching harvest maturity while the branches and stems remain green, are still likely to be physiologically active and engaged in plant defense response (Miklas et al., 2004).
QTL on Linkage Group B3
A QTL for partial resistance to white mold identified on linkage group B3, nearest RAPD marker K10.350, was expressed in two environments explaining 5.3% of the variation for disease score for Paterson in 2001 and 15.7% for Hatton (Table 1). Disease avoidance and physiological mechanisms likely underlie the partial resistance expressed by this QTL, because the same genomic region was associated with canopy porosity across all environments, canopy height in two environments, stay green stem trait in two environments, and lodging in one environment. In addition, the same genomic region was associated with harvest maturity in all environments and yield in two environments. The genomic region for this QTL is more than 20 cM from the minor-effect QTL for resistance to white mold located on B3 near RAPD marker G03.1150 (Park et al., 2001).
The ND88–106–04 allele for the B3 QTL, as defined by K10.350 RAPD marker, contributed favorably to reduced disease severity, increased plant height, and increased green stem trait. Conversely, the Aztec allele contributed favorably to increased yield, earlier maturity, and a more open canopy. Composite interval mapping depicts a 19 cM span of the genome which influences disease avoidance (open canopy) from the Aztec parent near K10.350 marker and physiological resistance (stay green stem) from the ND88–106–04 parent near AT7.470 marker (Fig. 3 ). For this genomic region, the challenge for breeders will be to select progeny which recombine favorable traits contributed by each parent in coupling-phase linkage.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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The RAPD markers, 015.1800 and BC20.1800, identified previously (Kolkman and Kelly, 2003; Ender and Kelly, 2005), and the AFLP 10 (EAGTMCTT.300) identified herein, provide initial markers for testing the effectiveness of marker-assisted breeding for the B2 QTL to improve white mold resistance. It may be worthwhile to backcross the B2 QTL into a susceptible background using marker-assisted selection (MAS) to validate the effect of the QTL in absence of other resistance factors, validate the effectiveness of MAS for the QTL itself, and to generate near-isogenic inbred lines for eventual analysis of candidate genes underlying the QTL. Eventually, those markers found to be tightly linked with the B2 QTL should be converted to SCAR markers to facilitate use by different programs.
The B3 QTL for partial resistance, as determined by regression analysis, had a minor effect in two environments. Composite interval mapping determined the QTL did not have a main effect; and, to date, this same B3 QTL has not been detected in any other populations. These results for B3 QTL suggest further research would be required to validate the importance of this genomic region in breeding for resistance to white mold disease in common bean.
Received for publication June 22, 2006.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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This article has been cited by other articles:
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  P. N. Miklas Marker-Assisted Backcrossing QTL for Partial Resistance to Sclerotinia White Mold in Dry Bean Crop Sci., May 31, 2007; 47(3): 935 - 942. [Abstract] [Full Text] [PDF]
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