| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
USDA-ARS, Forage and Vegetable Crop Research Unit, 24106 North Bunn Rd., Prosser, WA, 99350. This research was supported by the National ARS Sclerotinia Initiative
* Corresponding author (pmiklas{at}pars.ars.usda.gov).
 |
ABSTRACT |
|---|
 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Abbreviations: DAP, days after planting • QTL, quantitative trait loci • Pop-I, Population I • Pop-II, Population II • Pop-III, Population III • Pop-IV, Population IV • RAPD, random amplified polymorphic DNA • SCAR sequence-characterized amplified region.
|
INTRODUCTION |
|---|
|
TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
The recent identification of more than 10 independent quantitative trait loci (QTL) conditioning resistance (Miklas et al., 2001, 2003, 2007; Park et al., 2001; Kolkman and Kelly, 2003; Ender and Kelly, 2005) affirms the complexity and quantitative nature of white mold resistance in common bean. Those QTL identified with stable expression across environments and with relatively major effect (>10% of phenotypic variation explained), provide an opportunity for marker-assisted breeding to expedite development of cultivars with enhanced levels of white mold resistance. Two such QTL, with major effect and expression across multiple environments, were chosen for marker-assisted selection in this study.
A QTL derived from G122 resides on linkage group B7 near the Phs (phaseolin) seed protein locus and explained 38% of the phenotypic variation for disease reaction in the straw test and 26% in the field (Miklas et al., 2001). A QTL derived from NY6020-4 resides on linkage group B8 near the Co-4 locus for resistance to anthracnose caused by Colletotrichum lindemuthianum (Miklas et al., 2006b) and explained 35% of the variation in the straw test and 15% in the field (Miklas et al., 2003). The partial resistance conferred by these two QTL is likely conditioned by physiological mechanisms versus disease avoidance traits because they exhibit greater expression in the greenhouse straw test (Petzoldt and Dickson, 1996) than in field trials.
Other QTL derived from G122 on linkage group B1 and from NY6020-4 on B6 had relatively minor effect or no expression in greenhouse tests and were conditioned by disease avoidance traits in the field due to more open and porous canopy, increased plant height, and/or reduced lodging. Upright architecture promotes air and sunlight penetration into the plant canopy, creating a drier microclimate less conducive to white mold epidemics (Schwartz et al., 1987).
Pinto and great northern dry bean market classes grown across the United States lack partial physiological resistance to white mold and are severely affected by the disease. The purpose of this study was to determine whether marker-assisted selection for the QTL on B7 from G122 and QTL on B8 from NY6020-4 could be used effectively to transfer partial resistance to white mold into the susceptible pinto and great northern bean market classes.
|
MATERIALS AND METHODS |
|---|
|
TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
An F2 population from a three-way cross CO8112034//G122/Winchester was obtained from Syngenta Seed Company (Nampa, ID). An F2:4 line from this population with high level of resistance to white mold, as detected by the greenhouse straw test (Petzoldt and Dickson, 1996), was obtained. This line possessed the Phs SCAR (sequence-characterized amplified region) marker (described below) linked with the B7 QTL for partial resistance to white mold from G122 (Miklas et al., 2001). The F2:4 line, simulating a BC1F2:4 line because it contained two doses of pinto bean as recurrent parent, was backcrossed twice more to Winchester pinto bean. A BC2F1 with T-allele of the Phs marker was used as the parent for the subsequent backcross to Winchester. From the final backcross, two separate BC3F1 plants [Winchester*2/F2:4 line (CO8112034//G122/Winchester)] assayed for presence of the B7 QTL-linked SCAR marker were selfed. Fifty and 38 BC3F2 plants were advanced by single-seed descent for two generations followed by one generation of bulk seed increase to obtain 50 and 38 BC3F4:6 derived lines representing Population I (Pop-I) and Population II (Pop-II), respectively.
Population III (Pop-III) was derived from the pedigree Maverick/3/OT9630-17-5//NY6020-4/OT9630-17-5. A BC1F1 plant with the B8 QTL-linked markers (AW9.1200 RAPD and SS18.1650 SCAR; Miklas et al., 2003) was crossed with Maverick pinto. From the final backcross, a BC2F1 plant with the B8 QTL-linked markers was selfed to produce 33 F2 seed that were advanced by single-seed descent to produce 33 BC2F4:6 lines for Pop-III.
Population IV (Pop-IV) was derived from a conventional backcross with NY6020-4 as the donor parent of the B8 QTL, for partial physiological resistance to white mold, and Matterhorn great northern as the recurrent parent (Matterhorn*3/NY6020-4). A BC1F1 plant with B8 QTL-linked markers was backcrossed to the recurrent parent. A BC2F1 plant from the final backcross with the B8 QTL-linked markers was selfed to produce 41 BC2F2, which were advanced by single-seed descent to produce 41 BC2F4:6 lines for Pop-IV.
Greenhouse Straw Test
The straw test described by Petzoldt and Dickson (1996) was used to screen the BC2F4:6 or BC3F4:6 lines from each population for reaction to white mold. Each population was tested separately. An individual plant of each line represented a replicate, and there were six replicates randomized in complete blocks. The greenhouse environment was maintained at 18°C night to 25°C day with a 14-h daylength provided by sunlight and supplemental lighting. Plants were watered and fertilized for normal growth. The S. sclerotiorum culture T001.1, hyphal tip isolated from a sclerotia collected from ‘Newport’ navy bean in Quincy, WA in 1996, was the source of inoculum. The intact main stem, freshly cut above or below the fifth node, was fitted with a plastic straw containing an agar plug of mycelium approximately 28 d after planting (DAP). Mycelium germinated from a single sclerotium placed on a PDA plate was subcultured to prepare plates of growing mycelium for the inoculations. The entire 15- x 100-mm Petri plate of growing mycelium was used as inoculum once the mycelium reached the periphery of the plate, approximately 3 d after subculturing. Eight to 10 d after inoculation, reaction to white mold was scored from 1 to 9, where 1 = no symptoms, 2 = invasion of the stem past the site of inoculation but not to the first node, 3 = invasion of the stem to the first node, 4 = invasion of the internode slightly past the first node, 5 = invasion to the middle of the internode, 6 = invasion to the second node, 7 = invasion slightly past the second node, 8 = invasion to the middle of the second internode and beyond, and 9 = total plant collapse. Parents and checks utilized in the greenhouse straw tests and field trials below included Winchester, G122, Maverick, Matterhorn, and ‘Montrose’ pinto (Brick et al., 2001).
Field Trials
The BC3F4:6 and BC2F4:6 lines, parents, and checks for Pop-I, Pop-II, and Pop-III were examined for disease reaction to S. sclerotiorum in the field in 2004 and 2005. Pop-IV was only tested in 2005. Each population was tested in a separate experiment. The experiments, planted 19 June 2004 and 16 June 2005, were conducted at Paterson, WA. The field plot at the USDA-ARS Cropping Systems Research Farm at Paterson has a history of uniform S. sclerotiorum disease in common bean and most recently has been used successfully to differentiate among recombinant-inbred lines for field reaction to white mold for QTL studies (Miklas et al., 2001, 2003). The soil is a Quincy sandy loam (mixed, mesic Typic Torripsamments).
For each field test, a randomized complete-block design with three replications was used, except for Pop-II in 2004, which had only two replications planted due to a seed shortage. A plot consisted of one row 3 m long. Plot rows were spaced 0.56 m apart. Planting density was 234848 seeds ha–1. To promote white mold disease, approximately 6.3 mm of water was applied in two applications approximately 12 h apart, by overhead center-pivot irrigation on a daily basis from the onset of flowering to late pod-fill. To maintain vigorous plant growth, nitrogen was foliar applied by chemigation at a rate of 22 kg ha–1 on a weekly basis for 8 wk from the early seedling growth stage (about 18 DAP) to mid pod-fill (about 74 DAP).
Disease reaction was scored both years from 1 to 9 based on combined incidence and severity of infection at physiological maturity, where 1 = no diseased plants, 2 = 1 to 20% diseased plants and/or 1 to 5% infected tissue, 3 = 20 to 30% diseased plants and/or 5 to 10% infected tissue, 4 = 30 to 40% diseased plants and/or 10 to 20% infected tissue, 5 = 40 to 50% diseased plants and/or 20 to 30% infected tissue, 6 = 50 to 60% diseased plants and/or 30 to 40% infected tissue, 7 = 60 to 70% diseased plants and/or 40 to 50% infected tissue, 8 = 70 to 80% infected plants and/or 50 to 60% infected tissue, and 9 = 80 to 100% diseased plants and/or 60 to 100% infected tissue. The plots were scored for lodging (1 to 9, where 1 = no lodging and 9 = >90% lodged) at physiological maturity in 2005 for Pop-I, Pop-II, and Pop-IV. Maturity was estimated in 2005 as the number of days after planting till harvest maturity (d). The entire plot was harvested to estimate seed yield (kg ha–1) in 2005 for Pop-I, Pop-II, and Pop-IV. Estimated seed yield for Pop-III was obtained in 2004 from a nondisease nursery used for seed increase on the WSU Roza experimental farm near Prosser, WA. Pop-I, Pop-II, and Pop-III had seed increased at this site in 2004 in single-row plots (3-m length with 0.56-m row spacing) with three replications. Seed weight (g 100 seeds–1) was measured from the nondisease nursery plots to estimate seed size for the lines from the three populations. Seed weight for Pop-IV was obtained from the 2005 disease nursery.
Marker Assays
Genomic DNA was extracted using the FastDNA Kit (Bio 101, Vista, CA) according to the manufacturer's instructions. The DNA sample, representing a line or parent, was extracted from the emerging trifoliate leaves collected (approximately 50 mg) from three separate plants macerated together. The procedure involved placing the leaf tissue in a microcentrifuge tube with DNA extraction buffer and homogenizing the tissue for two 30-s periods at an intensity setting of 5.0 in the FastPrep FP 120 homogenizer (BIO 101/Savant, Vista, CA). The DNA was bound to a matrix using a binding buffer, washed, and then eluted with 100 µL of sterile distilled water. The purified DNA was adjusted to 10 ng µl–1 using a fluorometer before all PCR reactions.
The codominant SCAR marker previously developed (Kami et al., 1995) for the phaseolin seed protein locus (Phs) was used to assay individual BC2F1 and BC3F1 plants, and the 50 (Pop-I) and 38 (Pop-II) BC3F4:5 lines for presence of the B7 QTL derived from G122 (Miklas et al., 2001). The SCAR marker for the T-phaseolin allele at the Phs locus amplifies three homoduplex bands 249, 264, and 285 nucleotides in length, which are in coupling phase linkage with the QTL (Fig. 1). The SCAR is actually a codominant marker as it amplifies two other bands 249 and 270 nucleotides in length, for the susceptible S-phaseolin allele at the Phs locus.
|
Two markers linked with the QTL on B8 were assayed (Miklas et al., 2003). The SS18.1650 SCAR and AW9.1200 (5'-ACTGGGTCGG-3') RAPD (random amplified polymorphic DNA) markers, 1.9 cM apart in the original Benton/NY6020-4 RIL mapping population, were assayed across the BC1F1 and BC2F1 individuals and the 33 and 41 BC2F4:5 lines for Pop-III and Pop-IV, respectively, for detection of the QTL on linkage group B8 derived from NY6020-4.
The PCR protocol for the SS18.1650 SCAR marker was same as for the Phs SCAR except that a 63°C annealing temperature was used. The forward 5'-CTGGCGAACTGTAC ATGCAACATAC-3' and reverse 5'-CTGGCGAACTGATTCATACATTTTG-3' primers used for the SS18.1650 SCAR were the same as those published by Miklas et al. (2003).
The RAPD protocol for amplifying the AW19.1200 marker consisted of 25-µL reactions containing 2 U Stoffel fragment DNA polymerase (Applied Biosystems, Foster City, CA), 1X 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 an initial cycle at 94°C for 2 min; three cycles at 94°C for 1 min, 32°C for 1 min, 72°C for 2 min; followed by 30 cycles of 94°C for 10 s, 37°C for 20 s, and 72°C for 2 min; with a final 5 min extension period at 72°C. Amplified products from the PCR reactions for the AW9.1200 and SS18.1650 markers were separated on 1.4% agarose gels containing ethidium bromide (0.5 µg mL–1) for 5 h at 3V cm–1 constant voltage. Resolution of the Phs marker was better in a 2% agarose gel run at 12 h at 3V cm–1.
Statistical Analysis
The experimental data were analyzed by a general linear model using PROC GLM (SAS Institute, 1987). Least-square means were computed for all traits. The effect of the QTL on disease reaction in the four backcross populations was obtained by regression of the marker (presence = 1, absence = 0) and mean disease scores from each line, similar to a one-way ANOVA procedure, using PROC GLM (SAS Institute, 1987). The effect or phenotypic variation for disease score explained by the QTL-linked marker(s) was estimated by R2 value, and significance of the QTL on disease score was determined by an F-test.
|
RESULTS AND DISCUSSION |
|---|
|
TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
|
|
The effect of selection for the B7 QTL on agronomic traits is presented in Table 2. On average, the BC3F4:6 lines with the T-Phs marker yielded 4 (Pop-I) and 9% (Pop-II) less than those lines without the marker, indicating a slight yield drag effect from selection for the marker. However, the populations overall had 21 (Pop-I) and 24% (Pop-II) less yield than the recurrent parent suggesting that genomic regions independent of the target T-Phs locus had not been fully recovered from Winchester pinto with only three backcrosses. It is imperative that residual donor genome be reduced as much as possible because few if any lines derived from intergene pool (Andean/Middle American) hybridizations attain the yield potential or commercial phenotype of the Middle American parent (Kornegay et al., 1992; Singh, 1995; Welsh et al., 1995). G122 is of Andean origin and Winchester is of Middle American origin.
The wide intergene pool cross and too few backcrosses that contributed to reduced yield also likely contributed to an increase in lodging, 32% for Pop-I overall and 17% for Pop-II, in comparison to the recurrent parent. Lodging is an undesirable trait in dry bean that promotes white mold disease. The BC3F4:6 lines with T-Phs marker, across Pop-I and Pop-II, respectively, had harvest maturity delayed by 1 and 3 d, and seed weight increased by 7 to 6%, compared to lines absent the marker, which suggests that selection for the donor T-Phs allele had some linkage drag effects on these traits also. The Phs locus was reported by Johnson et al. (1996) to affect from 18 to 33% of total seed weight depending on environment and population. Thus, when using Phs locus for MAS of the B7 QTL for partial physiological resistance to white mold derived from G122, it advisable to periodically monitor selected lines for commercial seed size.
The BC3F4:6 lines with T-Phs marker listed in Table 2 were selected for consistently low disease scores across tests and favorable agronomic characteristics such as higher yield, adequate seed size, reasonable maturity, and moderate lodging. These select lines will be further evaluated for disease reaction and agronomic performance to identify candidate pinto bean lines with partial resistance to white mold for official germplasm release and registration. The partially resistant lines 011A-35 from Pop-I and 011D-3 and 011-D-25 from Pop-II are noteworthy for their high yield potential under moderate white mold disease pressure. The low yields for Montrose pinto, an otherwise high-yielding pinto in the absence of white mold disease (Brick et al., 2001), emphasizes the devastating effect white mold can have on pinto bean yield. Winchester, the recurrent parent, maintains yield performance under white mold pressure because of avoidance traits (upright architecture, minimal lodging) and perhaps other factors like the stay green trait, which contributes to physiological resistance (Miklas et al., 2007). Plants with branches and stems that remain green as the pods reach harvest maturity are still likely to be physiologically active and engaged in plant defense response (Miklas et al., 2004). Data on stay green trait was not collected in this study.
Marker-Assisted Selection for the B8 QTL
The B8 QTL conditioning partial physiological resistance to white mold and derived from the snap bean breeding line NY6020-4, as detected by the AW19.1200 RAPD marker, had major expression in the pinto Pop-III and great northern Pop-IV backcross populations, explaining 37 and 25%, respectively, of the phenotypic variation for disease reaction in the greenhouse straw test (Table 1). Results for the B8 QTL are based on selection for the AW19.1200 RAPD marker because it is closer to the QTL than the SS18.1650 SCAR (Table 1), as also observed by Miklas et al. (2003) in the original Benton/NY6020-4 mapping population. The markers do not flank the QTL (Miklas et al., 2003), which is another reason for only including results for the AW19.1200 marker from here on.
The B8 QTL had less effect in field trials for Pop-III, with only 13% of the variation in disease severity score explained in 2004 and none in 2005. A similar disparity of expression of B8 QTL between environments, 35% greenhouse and 15% field, was observed in the original Benton/NY6020-4 mapping population (Miklas et al., 2003). The effect of a QTL conferring partial physiological resistance in the field may be influenced by many factors, as stated above. The higher field expression for the B8 QTL in Pop-IV (28%) in comparison with the greenhouse straw test (25%) may be due to an unknown genetic background effect contributed by the recurrent parent (Matterhorn great northern).
Disease severity for BC2F4:6 lines with the B8 QTL, as implied by presence of the AW9.1200 marker, was reduced by 14 (Pop-III, 2004) and 18% (Pop-IV) in the greenhouse tests compared to those lines absent the QTL-linked marker, and was reduced by 17 (Pop-III, 2004) and 19% (Pop-IV) in the field. Marker-assisted selection for the B8 QTL had no adverse affect on yield, seed weight, maturity, or lodging. In fact the BC2F4:6 lines in Pop-IV with the B8 QTL-linked marker yielded 8% more than those lines without the marker and had 4% larger seed size.
Maturity for Pop-III overall in comparison to the recurrent parent Maverick was delayed by 19 d in a seed increase plot in 2004 and by 6 d in the 2005 disease nursery (Table 2). Conversely, for Pop-IV, seed weight and yield were less, and lodging increased compared to the recurrent parent. These comparisons suggest that the wide cross and residual donor genome of the snap bean parent NY6020-4, apart from the genomic region flanking the B8 QTL, are inhibiting recovery of agronomic performance at the same level of the recurrent parent for both market classes. A few BC2F4:6 lines with better agronomic performance and higher levels of partial resistance across tests were selected for further evaluation. Line 006C-26 (Pop-III), with high yield potential, and line 029C-40 (Pop-IV), with early maturity and reduced lodging, represent good candidate lines for use as donor parents in subsequent crosses with elite pinto and great northern bean.
|
CONCLUSIONS |
|---|
|
TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
When the B7 and B8 QTL were introgressed separately into pinto and great northern backcross populations by marker-assisted selection, they explained a major portion of the phenotypic variation for disease reaction in the greenhouse test but explained less in the field. Field expression of the partial physiological resistance conferred by the QTL is confounded by disease avoidance traits and may be overcome by too severe disease pressure. The level of resistance conferred by the QTL was fairly consistent across the four backcross populations resulting in a 
15 to 20% reduction in disease severity or by a score of 1 on the 9-point scale (reduced from 6 to 5, 5 to 4, etc.) in the greenhouse and field tests alike.
Less negative linkage drag effects were observed for agronomic traits, such as harvest maturity, seed weight, and yield, with the B8 QTL. Besides linkage drag from selection of the QTL, other remnant donor genome from the exotic parental sources likely contributed to poor agronomic performance. Thus, further breeding and germplasm development will be needed to recover pinto bean and great northern beans possessing both B7 and B8 QTL for partial resistance to white mold with acceptable maturity, seed quality, and yield. Initially marker-assisted selection among segregating progeny from crosses between select lines from Pop-I and Pop-II with those from Pop-III and Pop-IV would facilitate combining two unique and independent sources of partial resistance to white mold in a pinto bean or great northern bean market type. Eventually marker-assisted selection will be useful for combining the B7 QTL from G122 and B8 QTL from NY6020-4 with other sources of resistance and disease avoidance traits to attain even higher levels of resistance to white mold in dry bean.
|
NOTES |
|---|
|
TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
Received for publication August 15, 2006.
|
REFERENCES |
|---|
|
TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  Y. S. Chung, M. E. Sass, and J. Nienhuis Validation of RAPD Markers for White Mold Resistance in Two Snap Bean Populations Based on Field and Greenhouse Evaluations Crop Sci., November 24, 2008; 48(6): 2265 - 2273. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
