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Genetic Mapping of QTLs Underlying Partial Resistance to Sclerotinia sclerotiorum in Soybean PI 391589A and PI 391589B

^a Dep. of Crop and Soil Sciences, Michigan State Univ., East Lansing, MI 48824
^b Dep. of Plant Pathology, The Ohio State Univ., Wooster, OH 44691-4096
^c Dep. of Horticulture and Crop Science, The Ohio State Univ., Columbus, OH 43210-1086

^* Corresponding author (wangdech{at}msu.edu).

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Soybean [Glycine max (L.) Merr.] PI 391589B, a selection from PI 391589A was recently identified as a new source of resistance to Sclerotinia sclerotiorum (Lib.) deBary, which causes Sclerotinia stem rot. The objective of this study was to identify the quantitative trait loci (QTLs) associated with resistance to S. sclerotiorum in PIs 391589A and 391589B. BC₁F_4:5 and BC₁F_4:6 populations from a cross of ‘Kottman’(2) x PI 391589A and a population of F₂-derived lines from a cross of PI 391589B x IA2053 were evaluated for resistance to S. sclerotiorum in the field and in the greenhouse from 2003 to 2005 and genotyped with simple sequence repeat markers. Single factor analysis identified 18 markers on nine linkage groups (LGs) significantly (P < 0.05) associated with resistance to S. sclerotiorum in the two populations. Four regions on LGs E, F, M, and O were significantly associated with the disease resistance in both populations. The four regions are between Satt411 (12.9 cM) and Satt369 (56.2 cM) on LG E, between Satt269 (11.4 cM) and AW186493 (21.0 cM) on LG F, between Satt463 (50.1 cM) and Satt323 (60.1 cM) on LG M, and between Satt581 (106.0 cM) and Satt153 (118.14 cM) on LG O on the soybean composite map developed by Song and others in 2004. Composite interval mapping identified seven QTLs (P < 0.10), each explaining 6.0 to 15.7% of the phenotypic variance. A QTL on LG M near marker Satt463 (50.1 cM) is unique to PI 391589A and B. Therefore, PIs 391589A and 391589B offer breeders a new allele for resistance to the disease.

Abbreviations: AUDPC, area under disease progress curve • BLUB, best linear unbiased predictor • CIM, composite interval mapping • CTAB, cetyltrimethyl ammonium bromide • DSI, disease severity index • LG, linkage group • LOD, log of odds • MIM, multiple interval mapping • PCR, polymerase chain reaction • PDA, potato dextrose agar • PI, plant introduction • QTL, quantitative trait locus • SSR, simple sequence repeat

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
SCLEROTINIA STEM ROT, also called white mold, is caused by the fungal pathogen Sclerotinia sclerotiorum (Lib.) deBary and affects a large number of crops, including soybean [Glycine max (L.) Merr.]. This disease occurs more frequently in the Upper Midwest region of the United States and southern Canada in high-yield soybean production systems during cool, wet summers (Grau et al., 2004; Hoffman et al., 1998). The infection process begins with colonization of the soybean flower petals by S. sclerotiorum ascospores. Infection then spreads to pods, nodes, and stems and may result in premature plant death (Grau, 1988). Symptoms of infection include wilting, dying plants, white tufts of mycelium on the stems, leaves, and pods, and sclerotia on and in stems. Sclerotia are often harvested inadvertently along with the seed and may contribute to reduced seed quality as well as a broader distribution of the pathogen (Danielson et al., 2004; Grau et al., 2004). From controlled yield loss studies, for every 10% increase in disease incidence, yield reductions averaged between 170 and 330 kg ha^–1 (Hoffman et al., 1998). When environmental conditions are conducive, Sclerotinia stem rot can cause as much yield loss as soybean cyst nematode (Heterodera glycines Ichinohe) and Phytophthora root and stem rot (Phytophthora sojae Kaufmann & Gerdemann) (Arahana et al., 2001; Grau et al., 2004).

Since environmental conditions in the field are difficult to control, the most effective way to manage Sclerotinia stem rot is through planting cultivars that are physiologically resistant to infection by S. sclerotiorum (Kurle et al., 2001). Several sources of partial resistance have been identified, albeit none have complete resistance to this pathogen (Hoffman et al., 2002; Kim et al., 1999). Kim et al. (1999) evaluated 18 soybean genotypes for resistance to S. sclerotiorum and found that ‘S19-90’, ‘A2506’, ‘Colfax’, and ‘Corsoy 79’ had the highest levels of resistance. Hoffman et al. (2002) tested 6520 plant introductions (PIs) and identified 68 PIs, including PI 391589B, with partial resistance to S. sclerotiorum in field evaluations. Environmental factors play a large role in Sclerotinia stem rot development, which has hindered efforts to assay for resistance (Plant Health Initiative, 2008). The association and consistency of a number of greenhouse and laboratory screening methods with the field response have been evaluated, including infested oat (Avena sativa L.) seed cotyledon, mycelial plug cotyledon, mycelial plug-excised leaf, mycelial plug-cut stem, mycelial plug petiole, and the response of detached leaves to oxalic acid (Kim et al., 2000; Wegulo et al., 1998). Both the infested oat seed cotyledon (Kim et al., 2000) and the mycelial plug-cut stem assay (Vuong et al., 2004; Wegulo et al., 1998), and more recently the drop mycelium method (Chen and Wang, 2005), were reported to have moderate correlations with the expression of resistance in the field.

Partial resistance in soybean to S. sclerotiorum is inherited as a quantitative trait (Kim and Diers, 2000). Quantitative trait loci (QTLs) associated with the trait were studied in a limited number of resistance sources using genetic markers. Eighteen hundred forty-nine markers were mapped in 20 linkage groups on the integrated soybean genetic map (Song et al., 2004). Some of these markers were used to locate QTLs underlying partial resistance to S. sclerotiorum. Kim and Diers (2000) identified three QTLs associated with resistance to S. sclerotiorum in the soybean cultivar S19-90. Arahana et al. (2001) identified 28 putative QTLs in soybean cultivars Corsoy 79, Dassel, DSR173, S19-90, Vinton 81, and Williams 82. Some of the previously identified QTLs were also associated with flowering time, internode length, and lodging, suggesting that these may be disease escape mechanisms and not physiological resistance (Kim and Diers, 2000).

Quantitative trait loci conferring disease resistance could vary among different sources of resistance, and identifying QTLs from other resistant sources could help us understand the relationship among resistance genes and facilitate combining different resistance genes into one cultivar. The objective of this study was to characterize the QTLs conferring resistance to S. sclerotiorum in PI 391589A and PI 391589B. Designations A and B in the PI collection indicate that a subline was isolated from the original seed sample and maintained (Nelson et al., 1987). PI 391589A and PI 391589B originate from China and have different maturities but also differ in flowering, lodging, and seed composition traits (Nelson et al., 1987).

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Mapping Populations
Two soybean mapping populations were developed. A population of 94 F₂-derived lines from a cross of soybean PI 391589B (partially resistant) x IA2053 (moderately susceptible) was developed in Michigan. F₂ plants were individually harvested to create F_2:3 lines. Each F_2:3 line was advanced to the F_2:4 generation by harvesting a single pod with three seeds from each plant and bulking the seeds of all plants of the F_2:3 line. F_2:5 lines were produced in the same way from the F_2:4 lines. In Ohio, a backcross (BC₁F_4:5 and BC₁F_4:6) population of 230 families was developed by crossing the parents Kottman (moderately susceptible) (St. Martin et al., 2001) x PI 391589A (partially resistant) and backcrossing once to Kottman (Dorrance, unpublished data; Hoffman et al., 2002). The backcrossing resulted in 13 BC₁F₁ plants that provided progeny for the population. The number of families derived from each BC₁F₁ plant ranged from 10 to 26. The population was advanced by single-seed descent to the F₄ generation. No selection was practiced at any time in either population. The seeds of PI 391589A and PI 391589B were provided by Dr. Randy Nelson at the USDA-ARS at Urbana, Illinois and the seeds of IA2053 were provided by Dr. Walter Fehr at Iowa State University.

Resistance to Sclerotinia sclerotiorum in PI 391589B
The F_2:3 and F_2:4 populations were tested for Sclerotinia stem rot resistance by field inoculations in 2003 and 2004. Field experiments were performed at the Agronomy Farm of the Department of Crop and Soil Sciences at Michigan State University (East Lansing, MI). A randomized complete block design was used with three replications of the parental genotypes and the 94 F_2:3 lines in 2003 and six replications of the parental genotypes and the 94 F_2:4 lines in 2004. Each plot had one 3-m-long row of approximately 30 plants; rows were spaced 38 cm apart. After each inoculation the fields were sprinkler irrigated with approximately 2.5 mm of water every evening for 3 wk.

The F_2:3 population was planted on 28 May 2003 and was tested for Sclerotinia stem rot resistance by the cut-petiole inoculation method described by Del Rio et al. (2001). The S. sclerotiorum isolate HT 105, provided by Dr. Glen Hartman, USDA-ARS at Urbana, IL, was used as the inoculum. The HT 105 isolate was maintained by subculturing every 2 wk on potato dextrose agar (PDA) medium. To prepare inocula for field experiments, a plug of mycelia was transferred to PDA medium in 120- by 30-mm petri dishes maintained on a laboratory bench at room temperature (22–25°C) until the mycelia reached the edge of the plate in approximately 3 d. At that time a 1-cm diam., 8-mm-deep plug of mycelium was removed using an inverted 1-mL pipette filter tip (USA Scientific, Ocala, FL) and used to inoculate a soybean plant in the field. The mycelium plugs loaded in 1-mL pipette tips were prepared in the early morning of the inoculation day and kept on ice until placed on the plants. The second-youngest fully opened trifoliolate leaf was removed with a razor blade approximately 5 cm above the point of attachment to the petiole. The cut petiole was forced into the wide end of the pipette tip containing the mycelial plug to assure contact of the cut-petiole surface with the mycelia. Thirty plants of each line (>2700 plants for the entire population) were inoculated in each replication. The total number of inoculations of three replications exceeded our capacity of inoculations in a single day. Therefore, each replication was inoculated on a separate day, 13 August, 22 August, and 29 August, respectively. Most plants were in R5 growth stage (Fehr and Caviness, 1977) when they were inoculated. Plants were observed daily after inoculation until the first plant showed apical wilting symptoms. Plants were subsequently evaluated for wilting twice a week for the first 7 wk after each inoculation. The wilted apical parts were removed after data collection to prevent plant death to allow for F_2:4 seed production. The area under disease progress curve (AUDPC) was calculated for each line according to the formula (Shaner and Finney, 1977):

where y_i is percentage infection at time t_i.

The F_2:4 population was planted on 8 June 2004. To expedite field inoculations, a new method, the drop-mycelium method (Chen and Wang, 2005), was used. Sclerotinia sclerotiorum HT 105 mycelia were cultured in PDA medium for 3 d at room temperature (22–25°C). Twenty 8-mm diam. plugs of S. sclerotiorum HT 105 mycelia were put into 2 L of autoclaved liquid potato dextrose broth, and cultured for 5 d at room temperature with shaking at 100 rpm. The mycelial culture was blended for 10 s at high speed using a 1350-mL household blender (BlenderMaster, Hamilton Beach/Proctor-Silex, Mexico) to create a uniform suspension and was then transferred to 500-mL wash bottles. Approximately 3 mL of the mycelial suspension (sufficient to allow runoff) was applied to the apical meristems and the youngest one or two axillary meristems of each plant. Inoculation of the six replicate blocks was made on different days because our capacity did not allow us to inoculate all six blocks in a single day. The inoculations were performed on 10, 13, 17, 19, 23, and 24 August, when the plants were in R3 to R4 growth stages (Fehr and Caviness, 1977). The inoculation dates were chosen to avoid rainy days for the concern of the inoculum being washed off by the rain. When the majority of the plants reached physiological maturity (R7) (Fehr and Caviness, 1977), all inoculated plants in each row were individually rated for disease severity using the rating system of Grau et al. (1982), where 0 = no symptom, 1 = lesions on lateral branches only, 2 = lesions on the main stem but little or no effects on pod fill, and 3 = lesions on main stem resulting in poor pod fill or plant death. A disease severity index (DSI) was calculated for each line using the formula DSI = 100(r/3n) (where r = rating of each plant, n = number of plants rated, and 3n is the upper limit of the sum of ratings). The F_2:5 population was tested by greenhouse inoculation in 2005 using a randomized complete block design with three replications. Each replication consisted of two pots with approximately 10 plants. To facilitate infection, an inoculation chamber was made by covering a 5- by 1.5- by 1.5-m section of bench with plastic. Two misters were placed at each end of the plastic chamber. The plants were grown in the greenhouse outside the plastic chamber for about 3 wk (between V2 and V3 stages) and then transferred to the inoculation chamber. Inoculation was done using the drop-mycelium method as described by Chen and Wang (2005). Approximately 0.5 mL of the mycelial suspension was applied to the apical meristems. After each inoculation, the plastic chamber was covered and the misters emitted water mist for 1 min every 5 min. The growth chamber held one replication of the parental genotypes and 94 F_2:5 lines. Three replications were sequentially done in March and April 2005. The plants were scored for mortality on Day 5, 8, and 12 after inoculation. The AUDPC was calculated for each line according to the formula of Shaner and Finney (1977).

Resistance to Sclerotinia sclerotiorum in PI 391589A
The infested oat seed cotyledon assay and the cut stem assay were used to evaluate the population for Sclerotinia stem rot resistance. The infested oat seed cotyledon assay was similar to that described previously (Kim et al., 2000; Dorrance, 2003). Inoculum was produced on autoclaved oat grains in which 500 mL of oat grains were soaked overnight with 100 mL of dH₂0 in 2-L flasks and autoclaved on 2 d successively for 1 h. Autoclaved oats were seeded with 3- to 5-mm colonized agar plugs from a 2-d-old colony of S. sclerotiorum. Flasks were shaken daily for 2 wk, and the colonized oats were then dried on an air bench and stored at 4°C until used. For this assay, 12 to 15 plants per BC₁F_4:5 and BC₁F_4:6 family were planted in a 4-cm square pot in course vermiculate (Therm-O-Rock East, New Eagle, PA). Approximately 2 wk later, when the unifoliolate leaves began to emerge, the plants were inoculated with S. sclerotiorum. For each plant, a hole approximately 3 mm in size was made with a hole punch in one cotyledon in the bottom third closest to the stem, and a colonized oat kernel was placed in the hole. Plants were placed in a mist chamber for 48 h at 20°C and then removed from the mist chamber and placed in the greenhouse for 24 to 48 h. To determine a percentage of killed seedlings, disease data were collected on the number of dead plants and the total inoculated when the control plants (Williams 82) were 70 to 90% dead. Families were treated as entries; for every 15 entries, two controls were also inoculated. The experiment was planted as an incomplete block design with three replications in time for a total of 36 to 45 families assayed at each given time. Each incomplete block contained the checks and parents and 78 to 104 entries from Kottman(2) x PI 391589A.

The cut stem assay was similar to that described by Vuong et al. (2004). Seven seeds from each BC₁F_4:5 family were planted in the greenhouse in 15-cm plastic pots containing a 1:1:1 v/v soil mix of potting soil:perlite:sand. After 14 d, the number of seedlings per pot was thinned to five. After approximately 4 wk, when the plants reached the V5 or V6 growth stage (Pederson, 2004), the stem of each plant was cut horizontally 0.5 cm above the fourth or fifth node. A single mycelial plug from a 2-d-old S. sclerotiorum culture grown on PDA was placed with the colony side next to the stem of each plant. The inoculated plants were kept in the greenhouse under a cover of black mesh cloth for 48 h. Ambient temperatures during each experiment were 20°C for the first 48 h after inoculation and then 25°C until the experiment was completed. Lesions were measured 14 d after inoculation. Measurements were taken in millimeters from the cut stem to the lesion edge. The design was a randomized block design with four replications. The experimental unit was one pot with three plants in each replicate.

Population Genotyping and Marker Linkage Analysis
The PI 391589B population genotyping was performed on the F₂ generation in Michigan. Unopened trifoliolate leaves were collected from each F₂ plant in the field and kept on ice before the samples were transported to the laboratory. The leaf samples were kept at –80°C for at least 48 h and then lyophilized for approximately 72 h. DNA was extracted from the dried leaf tissues using a modified cetyltrimethyl ammonium bromide CTAB method (Kisha et al., 1997). In Ohio, with the PI 391589A population, DNA was extracted from dried tissue of the BC₁F_4:5 using the CTAB method (Saghai Maroof et al., 1984).

Single locus simple sequence repeat (SSR) markers which have been confirmed in numerous mapping populations (Cregan et al., 1999; Song et al., 2004), were used to genotype all of the populations. The SSR markers were Class I microsatellite markers with 2 to 3 bp SSR of >10 bp iterations. The SSR primer sequences were obtained from the SoyBase database (http://soybase.org/). The SSR primers were synthesized by Sigma Aldrich (St. Louis, MO). A total of 67 and 109 SSR markers were used to genotype PI391589A and PI 391589B populations, respectively, with an emphasis placed on regions previously reported to contain QTLs associated with S. sclerotiorum resistance (Arahana et al., 2001; Kim and Diers, 2000). In Michigan, polymerase chain reactions (PCRs) were run as previously described (Cornelious et al., 2005) and PCR products were analyzed in a 6% nondenaturing polyacrylamide gel system (Wang et al., 2003). In Ohio, reactions were run as previously described (Cregan et al., 1999), and PCR products (20 µL) were resolved on 4.5% high-resolution agarose gels (Amresco, Solon, OH), stained with ethidium bromide and visualized on a UV lightbox.

The computer program JoinMap 3.0 (Van Ooijen and Voorrips, 2001) and the Kosambi mapping function (Kosambi, 1944) were used to determine the linkage relationships among the polymorphic SSR markers for both populations. The grouping log of odds (LOD) threshold was set to 3.0 to divide markers into linkage groups. The marker order and positions within each linkage group were determined with a minimum LOD of 3.0 and a maximum recombination frequency of 0.35. The Kosambi mapping function was used to convert recombination frequencies to map distances. The linkage map resulting from JoinMap analysis was used as the map input in the QTL analysis.

Data Analysis and QTL Mapping
PI 391589A Population
The two generations of the PI 391589A population (BC₁F_4:5 and BC₁F_4:6) were analyzed separately. A mixed models analysis was used for the cotyledon assay to obtain the best linear unbiased predictor (BLUP) for each family (Stroup, 1989). The model for both generations was

where µ is overall mean, S_i is the effect of the ith incomplete block (F_4:5) or complete block (F_4:6), C_j is class of entry (j = 0 for recombinant line, and j = 1, 2, 3, and 4 for the four parents and checks), G(C)_jk is genotype within class for recombinant lines only (genotypic variance), SC_ij is block x class interaction, SG(C)_ijk is block x genotype interaction within class (experimental error), and _ijkl refers to sampling variation from plant to plant within an experimental unit. Class of entry was assumed to be a fixed effect, and all other terms were assumed to be random. This model permitted an analysis in which checks and parents were fixed effects and recombinant lines were random. Variance components were estimated using restricted maximum likelihood. Heritability, on a family mean basis, was calculated as _g²/_p², where _g² is genetic variance and _p² is phenotypic variance. The main effects of genotype, replicate, and day of measurement (7 and 14) on Sclerotinia lesion length were determined by ANOVA using SAS PROC GLM (SAS Institute, Cary, NC). For QTL detection, an ANOVA was run of the SSR marker data and BLUP severity values, and mean lesion lengths were analyzed as dependent variables. The model used for QTL detection was y_ij = µ + _i + _ij where y_ij is the phenotypic classification of the jth genotype of the ith marker class, µ is the population mean, _i is the effect of the ith marker class and _ij is the experimental error. The percent phenotypic variation in lesion length and disease severity explained by each SSR marker was calculated using the restricted maximum likelihood method of Proc Varcomp in SAS (SAS Institute, Cary, NC).

Linkage groups identified by single-factor analysis as containing QTLs were scanned at 5-cM intervals using LOD thresholds corresponding to a genomewide error rate of 5% estimated by 1000 permutations of the data (Churchill and Doerge, 1994) in the QTL analysis program MapQTL 4.0 (Van Ooijen et al., 2002). Marker cofactors were selected by the automatic cofactor selection option in MapQTL.

PI 391589B Population
The broad-sense heritability for Sclerotinia stem rot resistance for each year was calculated with the variance component method described by Fehr (1987). The variance components were estimated with PROC GLM of SAS (SAS Institute, Cary, NC) using the statistical model: Y_ij = µ + G_i + R_j + GR_ij + _ijk, where Y_ij is the observed phenotypic value of ith genotype (i = 1, ..., 94) in jth replication (j = 1, ..., 3 or 6), µ is the overall mean, G_i is effect of genotype, R_j is the effect of replication, GR_ij is the interaction of genotype by replication, and _ijk is the plant-to-plant variation within a plot. The components of variance were estimated on a plot basis. Pearson correlations among data collected in different years were determined with the PROC CORR of SAS.

Both the single marker analysis and the composite interval mapping (CIM) methods in WinQTLcart Version 2.5 (Wang et al., 2005) were used in the QTL analysis. For the CIM analysis, the number of control markers was set to five and the window size was set to 10.0 cM. The forward and backward regression method was used to select control markers. The threshold used to declare a QTL significant in CIM analysis was chosen to be genomewise P 0.10, which was obtained from 1000 permutations (Churchill and Doerge, 1994). The corresponding P value of a LOD score was obtained from the permutation results. The estimated QTL position was at the position on the linkage map where the LOD score peaks. All significant QTLs identified for a trait in the CIM analysis were analyzed together with the multiple interval mapping (MIM) method in WinQTLCart version 2.5 (Wang et al., 2005) to determine QTL interactions and the combined effects of all QTLs. MapChart 2.1 (Voorrips, 2002) was used to create the QTL LOD plots.

	RESULTS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Resistance to Sclerotinia sclerotiorum in PI 391589A
For both generations, we found significant differences among the families for the BLUP values calculated from the disease rating from the infested oat seed cotyledon assay. In addition, the families were continuously distributed for the reaction to S. sclerotiorum using BLUP values (Fig. 1 ). The mean percentage of dead plants per family for the entire population was 70%, with the controls Williams 82 81%, Kottman 83%, and S-1990 29%; PI 393589A had poor germination, and the number of plants that were inoculated was too low to include in the analysis. Mean lesion length values from the cut stem assay were also continuously distributed in the BC₁F_4:5 population for the greenhouse cut stem assay (Fig. 2 ). The population mean lesion length was 51.6 mm, and the values ranged from 29 to 83 mm. There was transgressive segregation in the population based on mean lesion length from the cut stem assay, albeit only the most susceptible lines differed significantly from the susceptible parent. The resistant parent, PI 391589A, had a mean lesion length of 41 mm, and the susceptible parent, Kottman, had a mean lesion length of 43 mm at 14 d after inoculation. The resistant and susceptible checks lesion lengths were 44 and 48 mm for S-1990 and Willams-82, respectively.

View larger version (18K): [in this window] [in a new window]   Figure 1. Distribution of best linear unbiased predictor (BLUP) values for (A) a BC1F4:5 and (B) a BC1F4:6 populations from a cross of ‘Kottman’ x PI 391589A from an infested oat-cotyledon greenhouse assay to evaluate for resistance to Sclerotinia sclerotiorum.

View larger version (15K): [in this window] [in a new window]   Figure 2. Distribution of mean lesion lengths for a BC1F4:5 population of ‘Kottman’ x PI 391589A for the cut stem assay to measure resistance to Sclerotinia sclerotiorum.

Resistance to Sclerotinia sclerotiorum, PI 391589B
Plant apical wilting was observed in all lines inoculated with the cut-petiole method in the field in 2003. The AUDPC values of the population obtained in the 2003 field evaluation ranged from 4.4 to 32.7, with an average of 15.7. The average DSI values of lines in the population obtained from the 2004 field test ranged from 0.7 to 70.6, with an average of 11.2 (Fig. 3 ). In the 2005 greenhouse evaluation, most susceptible plants died within 12 d after inoculation, which was much shorter than the 7 wk observed for similar level of mortality in the 2003 field evaluation. Therefore, the AUDPC values obtained from the greenhouse evaluation were smaller than those obtained from the 2003 field evaluation. The AUDPC values of the population obtained in the 2005 greenhouse evaluation ranged from 2.1 to 7.9, with an average of 5.3. The resistant and susceptible parents fell into the expected ranges in both field evaluations, but they both appeared in the resistance range in the 2005 greenhouse evaluation. No lines were significantly more resistant than the resistant parent in all three years' evaluations. However, lines that were significantly more susceptible than the susceptible parent were observed in the 2003 and 2004 field evaluations. The phenotypic data from 2003 and 2004 field evaluations were significantly (P < 0.0002) correlated with a correlation coefficient of 0.37. The phenotypic data obtained in the 2005 greenhouse evaluation were significantly (P < 0.04) correlated with only data from the 2004 field evaluation with a correlation coefficient of 0.21.

View larger version (18K): [in this window] [in a new window]   Figure 3. Distributions of the area under disease progress curve values in the 2003 field evaluation and the 2005 greenhouse evaluation and the disease severity index values in the 2004 field evaluation of the 94 F2-derived lines from the cross PI 391589B x IA2053.

QTLs Associated with Resistance in the PI391589A Population
A total of 70 SSR markers were used to genotype the population. WE chose SSR markers to provide thorough-genome coverage with at least one marker per linkage group (Song et al., 2004) but placed emphasis on regions previously reported to contain QTLs associated with S. sclerotiorum resistance (Arahana et al., 2001; Kim and Diers, 2000). Linkage groups (LGs) B1, D2, H, and K had one or two SSR markers, while the remaining markers' total map distance was 1048 cM, with an average interval length of 16.1 cM.

Three SSR markers, Satt369 (LG E), Satt540 (LG M), and Satt581 (LG O), were significantly (P < 0.05) associated with mean lesion length (Table 1 ). Best linear unbiased predictor severity values for the infested oat seed cotyledon assay indicated that three SSR markers were significantly (P < 0.05) associated with resistance in the BC₁F_4:5, Satt212 (LG E), Satt138 (LG G) and Satt463 (LG M). The QTL on LG E for mean lesion length and the QTL on LG M for BLUP severity were also identified using CIM (Fig. 4E ). The proportion of phenotypic variance explained by the QTLs on LGs E and M was 6.0 and 9.0%, respectively. The remaining QTLs identified by single factor analysis fell below the significance threshold (LOD = 1.6).

View larger version (28K): [in this window] [in a new window]   Figure 4. Log-of-odds (LOD) plots of putative quantitative trait loci (QTLs) for Sclerotinia stem rot resistance using data from (A, solid line) 2003 field, (A, dashed line; B, C) 2004 field, and (F) 2005 greenhouse in Michigan for the PI 391589B x IA2053 population. The LOD thresholds for P 0.10, obtained from 1000 permutation with the 2003, 2004, and 2005 data were 3.0, 2.4, and 3.0, respectively (shown as dotted straight lines in A, B, C, and F). For Kottman x PI 391589A populations, the putative QTLs were based on (D) lesion length data and (E) severity data obtained in the greenhouse in Ohio. The permutation-determined LOD thresholds for P < 0.05 were 1.6 and are shown as dotted straight line (D and E). 1-LOD and 2-LOD support intervals of each QTL are marked by thick and thin bars respectively between the linkage map and the plot. Map distances are in centimorgans, and the linkage groups (LGs) are named according to Song et al. (2004).

QTLs Associated with Resistance in the PI 391589B Population
Of the 1132 SSR markers tested for polymorphism between the two parents, 109 were polymorphic. Eighty-one of the 109 polymorphic markers were placed into 23 linkage groups, which were segments of all linkage groups except B1, C2, H, and N on the integrated soybean linkage map (Song et al., 2004). The total map distance of the 23 linkage groups with the 81 SSR markers was 627.2 cM, with an average interval length of 10.8 cM.

Single marker analysis identified 7, 10, and 9 markers significantly (P 0.05) associated with the trait values obtained in 2003, 2004, and 2005, respectively (Table 2 ). Seven markers, AW186493, Satt149, Satt153, Satt212, Satt245, Satt411, and Satt419, were significant in 2 of the 3 yr; the other 12 markers were significant in 1 of the 3 yr only (Table 2). The resistant alleles of half of the significant markers were from PI 391589B; the other half were from IA2053 (Table 2). The PI 391589B allele of marker Satt245 was associated with the disease resistance in 2003 field evaluation but was associated with the disease susceptibility in 2005 greenhouse evaluation.

View this table: [in this window] [in a new window]   Table 2. Markers significantly (P 0.05) associated with resistance to Sclerotinia sclerotiorum in the population of PI 391589B x IA2053 based on the results of single marker analysis.

	DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The other QTL found on LG A2 had an overlapping 2-LOD supporting interval with the QTL located between Satt233 and Sat327 (Fig. 4A). The two QTLs may either be linked QTLs or the same QTL, which can only be determined with further studies.

Quantitative trait loci were identified on LG E contributing to resistance from both PI 391589A and PI 391589B near Satt212. Previous studies by Arahana et al. (2001) identified the marker OP_m12 on LG E significantly associated with resistance to S. sclerotiorum. The QTL with resistant allele from PI 391589B was approximately 2.5 cM from marker Op_m12, based on the integrated soybean linkage map (Song et al., 2004); thus, this may be the same QTL. Satt212 was significantly associated with the resistance allele from PI 391589A, and Satt212 was at the LOD peak position of the QTL with resistant allele from PI 391589B (Fig. 4B). This confirms the existence of the QTL at this position.

Another QTL on LG E with the resistance allele from IA2053 is unique. However, this QTL was at the same map location as a QTL for resistance to soybean cyst nematode (Yue et al., 2001) and for resistance to peanut root-knot nematode [Meloidogyne arenaria (Neal) Chitwood] (Tamulonis et al., 1997). Arahana et al. (2001) also found that putative QTLs for resistance to S. sclerotiorum occurred in map regions containing resistance genes for other diseases such as sudden death syndrome (Fusarium virguliforme Aoki, O'Donnell, Homma, and Lattanzi), Phytophthora root and stem rot, and powdery mildew (Microsphaera diffusa Cooke & Peck). The colocalized QTLs for resistance to different pathogens are either closely linked independent QTLs or shared QTLs with pleiotropic effects. Further studies are needed to determine the relationship of these colocalized QTLs. A QTL on LG F for resistance to S. sclerotiorum from PI 391589B is also unique, and there are no previous reports of QTLs associated with disease resistance in this genomic region.

Three markers, Satt050 on LG A1 and Satt323 on LG M from Kottman and Satt394 on LG G from PI 391589A were significantly (P < 0.01) associated with resistance in the BC₁F_4:6 but not the BC₁F_4:5 in the cotyledon assay (Table 1). Resistance in the BC₁F_4:5 was significantly associated (P < 0.05) with Satt212 on LG E and Satt138 on LG G from PI391589A and with Satt463 on LG M from Kottman. Quantitative trait loci on LGs G and O were identified previously from five mapping populations developed by crossing resistant parents with a common susceptible parent (Arahana et al., 2001). The QTL on G, designated Sclero 4-5, contributed by the resistant parent DSR173, is in an interval within 15 cM of Satt138 (Arahana et al., 2001; Grant and Shoemaker, 2008). The QTL on LG M, linked to SSR markers Satt463 and Satt540, was also identified in PI 391589B with the marker Satt245, which is located between marker Satt463 and Satt540 on the integrated linkage map. This QTL appears to be unique to PI 391589A and PI 391589B. However, environmental factors that influence this expression or epistatic effects between parents may be important for this region as resistance was attributed to the susceptible parent in both populations dependent on the generation or disease assay.

Of the five QTLs detected in the PI 391589B x IA2053 population with the CIM analysis, three had resistance alleles from the susceptible IA2053. Four QTLs also had resistance from Kottman in the PI391589A population. Arahana et al. (2001) also identified many QTLs with resistant alleles from the susceptible parent, Williams 82, of their five mapping populations. In their study, Williams 82 had resistant alleles for 16 of the 28 QTLs for resistance to Sclerotinia stem rot. Over half of the QTLs for resistance were identified from the susceptible parents in both our study and the study by Arahana et al. (2001), suggesting that elite and susceptible cultivars may be good sources of resistance to the disease. More QTL mapping studies are needed to identify resistant alleles from susceptible cultivars.

Several different inoculation methods were used in this study. The cut petiole, cut stem, and cotyledon assays allowed for easier pathogen infection due to the lack of an epidermal barrier from the host plant. However, these methods could detect only the physiological resistance of the host plant. The drop-mycelium method allowed pathogen infection without wounding the plants before inoculation. Therefore, it could detect the resistance both due to the epidermal barrier as well as the physiological factors of the host plant. Ontogenic expression of resistance in 12- to 15-d-old seedlings in the cotyledon assay compared with plants that are in the V4 to V5 growth stages in the cut stem assay may also account for differences in QTLs detected in the first backcross of Kottman x PI391589A population. This population was planted in the field during 2004 and 2005, but disease did not develop despite numerous inoculations accompanied by mist irrigation. Kim et al. (2000) and others have reported inconsistent field evaluations or lack of correlation between field and controlled environment evaluations of S. sclerotiorum resistance. With a detached leaf assay, Arahana et al. (2001) reported only 7 out of 28 putative QTLs that were detected in more than one population, which individually explained 4 to 10% of the phenotypic variation. Environmental factors, such as reduced light levels, have been shown to reduce the level of resistance expressed in some cultivars (Pennypacker and Risius, 1999). The lack of consistent results among mapping studies to detect QTLs for Sclerotinia resistance may be explained by significant genotype x environment interactions, existence of different QTLs for resistance in the different sources of resistance used in the QTL mapping studies, or differences in the genetic background that affect expression of the QTLs. Studies that evaluate the expression of these QTLs under different controlled environments and different genetic backgrounds are greatly needed.

Kim and Diers (2000) estimated the broad-sense heritability of Sclerotinia resistance at 0.59 in a population of 152 F₃-derived lines from S-1990 crossed with Williams 82. The broad-sense heritability ranged from 0.09 to 0.44 in this study. Lower heritability estimates in the BC₁ PI 391589A populations may be a reflection of the greenhouse assays but also that the parent Kottman used in the two respective crosses did not differ greatly in the resistance response to S. sclerotiorum from PI 391589A.

Kim and Diers (2000) identified three QTLs on LGs C2, K, and M for Sclerotinia stem rot resistance from a partially resistant variety S19-90. No polymorphic markers were found in the three QTL regions in our study. Arahana et al. (2001) identified QTLs on LGs D2, K, and L. No markers from these QTL regions were significantly (P 0.05) associated with resistance to S. sclerotiorum in this study. PI391589A, PI 391589B, IA2053, or Kottman may not carry the resistant alleles of these QTLs, or the level of resistance expressed by these QTLs may not have been significant enough to be detected under our evaluation conditions. The lack of a complete coverage of soybean genome may have resulted in failure to detect some QTLs for resistance to Sclerotinia stem rot. Nevertheless, the QTLs identified or confirmed in this study will help breeders to combine the favorable alleles of these QTLs with favorable alleles of QTLs identified in other resistance sources.

	ACKNOWLEDGMENTS

 
We thank Dr. Walter Fehr for providing the seeds for IA2053 and Dr. Randy Nelson for providing seeds of PI 391589A and PI 391589B. We would like to thank B. Bardall, B. James, and R. Berry for assistance with field and greenhouse experiments, and Glenn Mills for assistance with population development. Salaries and research support provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, and the Plant Breeding and Genetics Program at Michigan State University (http://www.hrt.msu.edu/pbgp/index.html). This research project was also supported by National Sclerotinia Initiative (http://www.whitemoldresearch.com/) and Ohio's Soybean Producers' check-off dollars through the Ohio Soybean Council.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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Received for publication December 4, 2007.

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