Abstract
Resistance to Phomopsis seed decay (PSD) in soybean (Glycine max [L.] Merr.) could provide dependable control of this important disease that affects seed quality. Studies have shown that single dominant genes that are allelomorphically different confer low levels of PSD in MO/PSD-0259 and PI 80837. The objectives of this research were to identify simple sequence repeat (SSR) markers linked to genes for PSD resistance in PI 80837 and MO/PSD-0259 and to associate the resistance genes to known linkage groups. Crosses were made between the PSD-susceptible cultivar Agripro 350 and each of the resistant lines MO/PSD-0259 and PI 80837. F2 populations from each cross were grown and inoculated in the field. Individual plant reactions were characterized by determining the levels of seed infection, and DNA of F2 plants was extracted for SSR analysis and mapping. F2 segregation data showed that different single dominant genes condition PSD resistance in MO/PSD-0259 and PI 80837. Resistance in PI 80837 was linked to Sat_177 (4.3 cM) and Sat_342 (15.8 cM) on molecular linkage group (MLG) B2. In MO/PSD-0259, resistance was linked to Sat_317 (5.9 cM) and Sat_120 (12.7 cM) on MLG F. These data support work by Berger and Minor (Berger RD, Minor HC. 1999. An restriction fragment length polymorphism (RFLP) marker associated with resistance to Phomopsis seed decay in soybean PI 417479. Crop Sci 39:800–805.) in which PSD resistance in PI 417479, the resistant parent used to develop MO/PSD-0259, was associated with RFLP marker A708 on MLG F. These SSR markers should be useful in selection for resistant genotypes in breeding programs.
Diaporthe phaseolorum (Cke. & Ell.) Sacc. var. sojae (Lehman) Wehm. and Phomopsis longicolla (Hobbs et al. 1985) (Diaporthe/Phomopsis complex [DPC]), which cause Phomopsis seed decay (PSD), are important fungal pathogens of soybean (Glycine max [L.] Merr.) seed worldwide (Kmetz et al. 1978, 1979; Hartman et al. 1999;). PSD begins when overwintering DPC conidia are rain splashed onto leaves and stems during periods of high moisture and warm temperatures. The spores then directly penetrate the tissue causing latent infections. During leaf and stem senescence when warm and moist conditions persist, the infected tissues become blighted with pycnidia oozing conidia that are disseminated to developing flowers and pods. Once pods become infected, the fungi can directly penetrate and colonize the seed coat causing a latent infection until seed maturation where infections give rise to seed decay or seed with reduced vigor and quality (Kmetz et al. 1978; McGee 1983; Rupe and Ferriss 1986; Rupe 1990; Mayhew and Caviness 1994).
Several cultivars and plant introductions (PI) have exhibited different levels of resistance to PSD. Of these, only PI 417479 and PI 80837 have been studied in some detail. Inheritance studies on PI 417479 suggested that resistance to PSD is conferred either by 1 dominant gene or 2 complimentary dominant genes (Zimmerman and Minor 1993). Restriction fragment length polymorphism (RFLP) analysis of an F2 population from AP 350 × PI 417479 indicated that PSD resistance was associated with marker A708 on molecular linkage group (MLG) F and with marker A162 on MLG H. These RFLP markers accounted for 62.2% and 4.5% of the phenotypic variation, respectively. Additionally, in an F2 population from PI 417479 × Williams 82, only RFLP marker A708 was significantly associated with PSD resistance, accounting for 21.0% of the phenotypic variation (Berger and Minor 1999). PSD resistance in PI 417479 was used to develop the resistant breeding line MO/PSD-0259 (PI562694) (Minor et al. 1993). Field evaluations in Missouri, Nebraska, and Arkansas showed that MO/PSD-0259 had low levels of PSD (0–6%) that were not different from PI 417479 (Elmore et al. 1998; Jackson et al. 2005). Recently, MO/PSD-0259 was crossed with the PSD-susceptible cultivar Agripro 350 (AP 350) to characterize the PSD resistance. Results indicated that resistance in MO/PSD-0259 is conditioned by a single dominant gene (Jackson et al. 2005).
Compared with PI 417479 and MO/PSD-0259, less is known about PSD resistance in PI 80837 (Yelen and Crittenden 1967; Roy and Abney 1988; Ploper et al. 1992; Jackson et al. 2005). Field studies in Indiana and Arkansas showed that PSD resistance in PI 80837 was consistent over different environments and years (Ploper et al. 1992; Jackson et al. 2005). Additionally, PI 80837 had low levels of pod and stem blight, caused by D. phaseolorum var. sojae and P. longicolla, whereas MO/PSD-0259 had severe blight on both stems and pods (Jackson 2000). Yelen and Crittenden (1967) argued that pod tissue in PI 80837 retarded infection by P. sojae, and Roy and Abney (1988) showed that unwounded pods of PI 80837 inoculated with P. longicolla had significantly less PSD than wounded pods. Because PSD resistance in PI 80837 appears to be different from resistance in MO/PSD-0259, inheritance studies were done to further characterize the genetics of resistance in PI 80837 and MO/PSD-0259. Results from multiple F2 populations and F2:3 lines over 2 years indicated that PSD resistance in PI 80837 and MO/PSD-0259 was conditioned by different single dominant genes (Jackson et al. 2005).
Several simple sequence repeat (SSR) markers have been identified on all 20 MGLs (Cregan et al. 1999; Shoemaker et al. 2004). These markers have been used as linkage tools, enabling mapping of several disease resistance genes including those for Soybean mosaic virus, soybean sudden death syndrome, and soybean cyst nematode (Meksem et al. 1999; Gore 2000; Iqbal et al. 2001; Koning et al. 2002; Qiu, 1999).
Genes for resistance to PSD in PI 80837 and MO/PSD-0259 are different and potentially useful for breeding resistant cultivars. Because SSR markers provided good tools for genetic linkage and mapping (Cregan et al. 1999; Shoemaker et al. 2004; Song et al. 2004) and for marker-assisted selection (MAS), the objectives of this study were to identify SSR markers linked to PSD-resistance genes in PI 80837 and MO/PSD-0259.
Materials and Methods
Plant Material
F1 hybrid seed were obtained from crosses between the cultivar AP 350 and each of the 2 soybean lines, PI 80837 and MO/PSD-0259. AP 350 is susceptible to PSD, whereas PI 80837 and MO/PSD-0259 are resistant to PSD (Roy and Abney 1988; Ploper et al. 1992; Minor et al. 1993; Zimmerman and Minor 1993; Jackson et al. 2004). F2 populations of 148 seed (AP 350 × PI 80837) and 140 seed (AP 350 × MO/PSD-0259) were produced from greenhouse grown F1 plants.
Assays of Resistance to PSD
Seeds of F2 populations from the crosses of AP 350 × MO/PSD-0259 and AP 350 × PI 80837 were planted as 2 separate tests in the field at Kibler, AR in 2003. Seeds were sown in rows with approximately 5 cm between each F2 seed. A single row of each parent (1.8 m) was planted every 25 to 30 F2 seed as checks. In both tests, rows were spaced 0.91 m apart, and border rows were planted on row ends and sides to maintain uniform environmental conditions.
To promote Phomopsis seed infection in the tests, 3 spray applications of P. longicolla conidial suspensions were made approximately 11 days apart starting when plants were at approximately R5 and ending at approximately R7 (Fehr et al. 1971). For inoculum, an isolate of P. longicolla previously tested for virulence on soybean in the field and greenhouse (Jackson 2000) with consistently good sporulation was used. Conidia were streaked onto Difco potato dextrose agar (PDA) and incubated at room temperature under fluorescent light with a 14-h photoperiod for 18–20 days. Cultures were flooded with sterile deionized water and agitated to disperse conidia. Conidial suspensions were adjusted to approximately 2.5 × 105 conidia/ml using a hemacytometer. Plants were sprayed with a backpack sprayer until pods were covered with drops of suspension. Inoculum was applied at dusk prior to dew or scheduled overhead irrigation to provide conditions favorable for infection.
Seed were collected from the lower 65 cm of parents and F2 plants 10 days after maturity (R8) using a single plant thresher. A random sample of 30 seed was taken from each parent and F2 plant. Samples were surface disinfested with 0.5% NaOCl amended with 5 drops of Tween 20/l and rinsed twice with sterile deionized water. Seed were plated (6 seeds/plate) on PDA amended after autoclaving with about 75 μg/ml streptomycin sulfate, 1μg/ml fenpropathrin (Valent USA Corp.) and acidified to pH 4.8 with lactic acid. Plates were incubated at room temperature under fluorescent light with a 14-h photoperiod for 10 days when the incidence (% seed infection) by Phomopsis was recorded.
Data Analyses and Resistance Classification
Percent Phomopsis seed infection of parent plants grown within each F2 population was analyzed by analysis of variance (P = 0.05; JMP, SAS Institute Inc., Cary, NC). Arcsine transformation was done before analyses and compared with analyses of the original percentage data. Because transformation did not change significance, untransformed percentage data were used. F2 plants were considered resistant to Phomopsis infection if they fell below the upper 95% confidence interval of the resistant parent plants for the respective population. Resistant plants were those having <29.6% PSD incidence for AP 350 × MO/PSD-0259 and <29.0% PSD incidence for AP 350 × PI 80837.
Chi-square analyses were used in all tests to determine the goodness of fit of the observed F2 data from both populations to the expected ratios for segregation of a single dominant gene.
DNA Isolation and SSR Analysis
Twenty-eight days after planting, parents and F2 plants from each population were labeled with plastic tags. Young leaf tissue was collected from each plant, frozen, lyophilized, ground into fine powder with liquid nitrogen, and then used for DNA isolation with a cetyl trimethyl ammonium bromide (CTAB) method. In brief, 500 μl of DNA extraction buffer containing 0.140 M sorbitol, 0.22 mM Tris–HCl, 0.022 M EDTA, 0.8 M NaCl, 0.8% CTAB, and 1.0% Sarcosine was added to tubes containing macerated leaf tissue. Tubes were incubated at 65 °C for 25 min then 300 μl of chloroform:isoamyl alcohol (24:1) was added, and the solution was mixed and centrifuged at 6000 × g for 25 min. The supernatant was collected, and an equal volume (1:1) of chilled 70% isopropyl alcohol was added to precipitate the DNA. Sterile pipette tips were used to remove DNA precipitants, which were then incubated in an RNase solution (1 μg/ml) to remove RNA. The DNA was washed with 70% ethanol, dried overnight, and resuspended in Tris EDTA. DNA concentration was determined for each sample by a spectrophotometer (A260 nm/A280 nm) and diluted to 20 ng/μl for polymerase chain reaction (PCR) reactions. Bulked segregant analysis (Michelmore et al. 1991) was performed on each population with SSR primers selected from all 20 MLG. The bulks were constructed with an equal amount of DNA from 10 resistant F2 plants and 10 susceptible F2 plants, respectively. PCR amplifications were run for 35 cycles at 94 °C for 30 s, 50 °C for 30 s, 72 °C for 60 s in a total volume 25 μl containing 16.6 μl deionized H2O, 2.5 μl 10× buffer (Promega, catalog number: M1901), 2 μl 25 mM Mg2+, 0.8 μl 10 mM dNTPs, 0.5 μl of each forward and reverse SSR primer (5 μM), 0.1 μl 5 U/μl Taq. (Promega, catalog number: M2668), DNA polymerase (Promega, catalog number: R2668), and 2 μl 20 ng/μl DNA template. The PCR products were separated on 3% superfine resolution (ARMESCO) agarose gels stained by ethidium bromide, and the images were recorded with a BioRad Image System (BioRad USA). Primers showing polymorphisms between the two parents and two corresponding bulked segregant lines were used to assess the individual genotype of plants in each population. Chi-square test was used to test the model for a single dominant gene. The software Mapmaker 3.0/EXP was used to calculate the genetic distances between SSR markers and soybean resistance genes and to draw linkage maps (Lander et al. 1987).
Results
SSR Markers and Genetic Mapping of the Phomopsis Seed Decay Resistance Gene in MO/PSD-0259 Soybean (Rpsd1)
One hundred and forty F2 plants from AP 350 × MO/PSD-0259 were used to characterize PSD resistance. Among them, 96 plants were resistant and 44 plants were susceptible. The ratio of resistant: susceptible plants fit a 3:1 (χ2 = 3.080, P = 0.10–0.05), indicating that PSD resistance is controlled by a single dominant gene (Table 1).
Phenotypic reactions to PSD of parents and an F2 population from AP 350 × MO/PSD-0259 inoculated with Phomopsis longicolla in the field and genotypes of F2 plants based on SSR markers
| Parents/populationa | Genotype | No. of plantsb | Chi-square 3:1 or 1:2:1 ratio | |||
| All R | H | All S | Value | Probability | ||
| AP 350 (S) | 1 | 15 | ||||
| MO/PSD-0259 (R) | 15 | 1 | ||||
| (AP 350 × MO/PSD-0259) F2 | 96 | 44 | 3.08 | 0.10–0.05 | ||
| Sat_317 | 29 | 59 | 37 | 1.28 | 0.75–0.50 | |
| Sat_120 | 33 | 53 | 39 | 2.99 | 0.25–0.10 | |
| Parents/populationa | Genotype | No. of plantsb | Chi-square 3:1 or 1:2:1 ratio | |||
| All R | H | All S | Value | Probability | ||
| AP 350 (S) | 1 | 15 | ||||
| MO/PSD-0259 (R) | 15 | 1 | ||||
| (AP 350 × MO/PSD-0259) F2 | 96 | 44 | 3.08 | 0.10–0.05 | ||
| Sat_317 | 29 | 59 | 37 | 1.28 | 0.75–0.50 | |
| Sat_120 | 33 | 53 | 39 | 2.99 | 0.25–0.10 | |
S = susceptible; R = resistant. Mean seed infection of AP 350 = 36.4% and MO/PSD-0259 = 21.4% based on 16 individual plants.
All R, all resistant; H, heterozygous; and All S, all susceptible.
Phenotypic reactions to PSD of parents and an F2 population from AP 350 × MO/PSD-0259 inoculated with Phomopsis longicolla in the field and genotypes of F2 plants based on SSR markers
| Parents/populationa | Genotype | No. of plantsb | Chi-square 3:1 or 1:2:1 ratio | |||
| All R | H | All S | Value | Probability | ||
| AP 350 (S) | 1 | 15 | ||||
| MO/PSD-0259 (R) | 15 | 1 | ||||
| (AP 350 × MO/PSD-0259) F2 | 96 | 44 | 3.08 | 0.10–0.05 | ||
| Sat_317 | 29 | 59 | 37 | 1.28 | 0.75–0.50 | |
| Sat_120 | 33 | 53 | 39 | 2.99 | 0.25–0.10 | |
| Parents/populationa | Genotype | No. of plantsb | Chi-square 3:1 or 1:2:1 ratio | |||
| All R | H | All S | Value | Probability | ||
| AP 350 (S) | 1 | 15 | ||||
| MO/PSD-0259 (R) | 15 | 1 | ||||
| (AP 350 × MO/PSD-0259) F2 | 96 | 44 | 3.08 | 0.10–0.05 | ||
| Sat_317 | 29 | 59 | 37 | 1.28 | 0.75–0.50 | |
| Sat_120 | 33 | 53 | 39 | 2.99 | 0.25–0.10 | |
S = susceptible; R = resistant. Mean seed infection of AP 350 = 36.4% and MO/PSD-0259 = 21.4% based on 16 individual plants.
All R, all resistant; H, heterozygous; and All S, all susceptible.
Because RFLP marker A708 on soybean MLG F was reported to be associated with the PSD resistance in PI 417479, the parent of MO/PSD-0259 (Berger and Minor 1999), 11 SSR primers were specifically selected from MLG F to screen the parents and 2 bulks for polymorphisms. Among the 11 markers screened, only Sat_317 and Sat_120 were found to be polymorphic and therefore were used to amplify DNA from 125 plants of the original F2 population of 140 plants.
Sat_317 produced a band of approximately 190 base pairs (bp) in the resistant parent and bulk and a band of approximately 210 bp in the susceptible parent and bulk (Figure 1). With primer Sat_317, 29 resistant plants produced the 190-bp band, whereas 35 susceptible plants produced the 210-bp band. Fifty-six resistant and 3 susceptible plants produced both bands, whereas 2 resistant plants produced the 210-bp band. Overall in this population, 29 plants had the 190-bp band (resistant), 37 plants had the 210-bp band (susceptible), and 59 plants had both bands (heterozygous). The ratio of resistant: heterozygous: susceptible genotypes fit a 1:2:1 (χ2 = 1.275, P = 0.75–0.50) (Table 1).
PCR profile of SSR markers Sat_317 and Sat_120 linked to PSD resistance in AP 350 × MO/PSD-0259 F2 population. L, 100-bp ladder; 1, susceptible parent; 2, resistant parent; 3, susceptible bulk (N = 10 plants); 4, resistant bulk (N = 10 plants); 5–11, resistant F2 plants; and 12–18, susceptible F2 plants.
Sat_120 produced a band of approximately 310 bp in the resistant parent and bulk and a band of approximately 360 bp in the susceptible parent and bulk (Figure 1). With primer Sat_120, 32 resistant plants produced the 310-bp band, and 36 susceptible plants produced the 360-bp band. Fifty resistant and 2 susceptible plants produced both bands, whereas 4 resistant plants produced the 360-bp band and 1 susceptible plant produced the 310-bp band. Overall in this population, there were 33 plants that had the 310-bp band, 39 plants that had the 360-bp band, and 53 plants that had both bands. The ratio of resistant: heterozygous: susceptible genotypes fit a 1:2:1 (χ2 = 2.986, P = 0.25–0.10) (Table 2).
Phenotypic reactions to Phomopsis seed infection of parents and an F2 population from AP 350 × PI 80837 inoculated with Phomopsis longicolla in the field and genotypes of F2 plants based on SSR markers
| Parents/populationa | Genotype | No. of plantsb | Chi-square 3:1 or 1:2:1 ratio | |||
| All R | H | All S | Value | Probability | ||
| AP 350 (S) | 0 | 17 | ||||
| PI 80837 (R) | 15 | 2 | ||||
| (AP 350 × PI 80837) F2 | 105 | 43 | 1.30 | 0.50–0.25 | ||
| Sat_177 | 37 | 68 | 41 | 0.90 | 0.75–0.50 | |
| Sat_342 | 40 | 66 | 40 | 1.36 | 0.75–0.50 | |
| Parents/populationa | Genotype | No. of plantsb | Chi-square 3:1 or 1:2:1 ratio | |||
| All R | H | All S | Value | Probability | ||
| AP 350 (S) | 0 | 17 | ||||
| PI 80837 (R) | 15 | 2 | ||||
| (AP 350 × PI 80837) F2 | 105 | 43 | 1.30 | 0.50–0.25 | ||
| Sat_177 | 37 | 68 | 41 | 0.90 | 0.75–0.50 | |
| Sat_342 | 40 | 66 | 40 | 1.36 | 0.75–0.50 | |
S = susceptible; R = resistant. Mean seed infection of AP 350 = 44.4% and PI 80837 = 23.1% based on 17 individual plants.
All R, all resistant; H, heterozygous; All S, all susceptible.
Phenotypic reactions to Phomopsis seed infection of parents and an F2 population from AP 350 × PI 80837 inoculated with Phomopsis longicolla in the field and genotypes of F2 plants based on SSR markers
| Parents/populationa | Genotype | No. of plantsb | Chi-square 3:1 or 1:2:1 ratio | |||
| All R | H | All S | Value | Probability | ||
| AP 350 (S) | 0 | 17 | ||||
| PI 80837 (R) | 15 | 2 | ||||
| (AP 350 × PI 80837) F2 | 105 | 43 | 1.30 | 0.50–0.25 | ||
| Sat_177 | 37 | 68 | 41 | 0.90 | 0.75–0.50 | |
| Sat_342 | 40 | 66 | 40 | 1.36 | 0.75–0.50 | |
| Parents/populationa | Genotype | No. of plantsb | Chi-square 3:1 or 1:2:1 ratio | |||
| All R | H | All S | Value | Probability | ||
| AP 350 (S) | 0 | 17 | ||||
| PI 80837 (R) | 15 | 2 | ||||
| (AP 350 × PI 80837) F2 | 105 | 43 | 1.30 | 0.50–0.25 | ||
| Sat_177 | 37 | 68 | 41 | 0.90 | 0.75–0.50 | |
| Sat_342 | 40 | 66 | 40 | 1.36 | 0.75–0.50 | |
S = susceptible; R = resistant. Mean seed infection of AP 350 = 44.4% and PI 80837 = 23.1% based on 17 individual plants.
All R, all resistant; H, heterozygous; All S, all susceptible.
The PSD-resistance gene (Rpsd1) in MO/PSD-0259 was mapped using the 2 SSR markers Sat_317 and Sat_120. SSR marker Sat_317 is 5.9 cM from the PSD-resistance gene (Rpsd1), whereas SSR marker Sat_120 is 12.7 cM from the resistance gene on the same side of MLG F (Figure 3).
SSR Markers and Genetic Mapping of the Phomopsis Seed Decay Resistance Gene in PI 80837 Soybean (Rpsd2)
One hundred and forty-eight F2 plants from AP 350 × PI 80837 were used to characterize PSD resistance in PI 80837. Among these, 105 plants were resistant, and 43 plants were susceptible. The ratio of resistant: susceptible plants fit a 3:1 (χ2 = 1.297, P = 0.50–0.25), indicating that PSD resistance is controlled by a single dominant gene (Table 2).
One hundred and sixty randomly selected SSR primers, covering all 20 MLGs of the soybean genome, were used to screen the parents of the (AP 350 × PI 80837) F2 population. Fifty primers produced polymorphisms between the parents and were used to screen resistant and susceptible bulks for polymorphisms. Only Sat_177, located on MLG B2, was found to be polymorphic and therefore was used to amplify DNA from 146 plants of the original F2 population of 148 plants.
Sat_177 produced a band of approximately 200 bp in the resistant parent and bulk and a band of approximately 180 bp in the susceptible parent and bulk (Figure 2). With primer Sat_177, 35 resistant plants produced the 200-bp band and 38 susceptible plants produced the 180-bp band. Sixty-seven resistant plants and 1 susceptible plant produced both bands, whereas 2 susceptible plants produced the 200-bp band and 3 resistant plants produced the 180-bp band. Overall in this population, 37 plants had the 200-bp band, 41 plants had the 180-bp band, and 68 plants had both bands. The ratio of resistant:heterozygous:susceptible genotypes fit a 1:2:1 (χ2 = 0.904, P = 0.75–0.50) (Table 2). Based on these data, SSR marker Sat_177 is located 4.3 cM from the PSD-resistance gene (Rpsd2) on MLG B2.
PCR profile of SSR markers Sat_177 and Sat_342 linked to PSD resistance in AP 350 × PI 80837 F2 population. L, 100-bp ladder; 1, susceptible parent; 2, resistant parent; 3, susceptible bulk (N = 10 plants); 4, resistant bulk (N = 10 plants); 5–11, resistant F2 plants; and 12–18, susceptible F2 plants.
Because SSR marker Sat_177 is on MLG B2, an additional 5 SSR primers, covering a 24-cM region around Sat_177, were selected and used to screen the parents and bulks from the (AP 350 × PI 80837) F2 population. Only Sat_342 was polymorphic and therefore used to amplify 146 plants in the F2 population.
Sat_342 produced a band of approximately 200 bp in the resistant parent and bulk and a band of approximately 220 bp in the susceptible parent and bulk (Figure 2). With primer Sat_342, 39 resistant plants produced the 200-bp band, and 32 susceptible plants produced the 220-bp band. Fifty-eight resistant plants and 8 susceptible plants produced both bands, whereas 1 susceptible plant produced the 200-bp band and 8 resistant plants produced the 220-bp band. Overall in this population, 40 plants had the 200-bp band, 40 plants had the 220-bp band, and 66 plants had both bands. The ratio of resistant:heterozygous:susceptible genotypes fit a 1:2:1 (χ2 = 1.355, P = 0.75–0.50) (Table 2). Based on these data, SSR marker Sat_342 is mapped at 15.8 cM from Rpsd2 on the same side as marker Sat_177 on MLG B2 (Figure 3).
Proposed genetic linkage maps of PSD resistance genes in soybean. Rpsd1 linkage map based on Sat_177 and Sat_342 on MGL B2 in PI 80837 and Rpsd2 linkage map based on Sat_317 and Sat_120 on MGL F in MO/PSD-0259. Both resistance genes were localized onto the IGLM (Cregan et al. 1999) based on linked SSR markers. Localization of different soybean resistance genes was based on previous reports.
Because Rpsd1 conferring PSD resistance in MO/PSD-0259 and Rspd2 conferring PSD resistance in PI 80837 are located on different MLGs (F and B2), we screened both MO/PSD-0259 and PI 80837 using markers associated with each resistance gene for polymorphisms. No polymorphisms were found between the genotypes (data not shown).
Discussion
In the current work, we have identified the map locations of 2 important genes controlling PSD in the MO/PSD-0259 and PI 80837 genetic backgrounds. It is important to point out that 1 plant of the 30 susceptible parental lines screened was scored as resistant in the study, whereas 1 plant of the MO/PSD-0259 and 2 plants of the PI 80837 resistant parental lines were scored as susceptible. The resistant scoring of the AP 350 plant was most likely due to escape, whereas the susceptible scoring of the resistant parental lines was probably due to pod wall disruptions caused by insects or other physical means.
In previous inheritance studies by Zimmerman and Minor (1993), PSD-resistance phenotypes from (PI 417479 × AP 350) and (PI 417479 × PI 91113) F2 populations at 1 field site fit a 3:1(R:S) model for a single dominant gene (Rollins Bottom site, P = 0.90–075), whereas the same populations fit a 9:7 (R:S) model for 2 complimentary dominant genes at a second field location (ARC site, P = 0.50–0.30). It was concluded that PSD resistance in PI 417479 was due to 2 complimentary genes (Zimmerman and Minor 1993; Minor et al. 1995). In 1999, Berger and Minor reported that resistant phenotypes from (PI 417479 × AP 350) and (PI 417479 × Williams 82) were associated with RFLP marker A708 on MLG F. Because PSD resistance in PI 417479 was used to develop MO/PSD-0259 (Minor et al. 1993), studies were done by Jackson et al. (2005) to characterize the PSD resistance in MOP/PSD-0259. They found that a single dominant gene conditions resistance. Genotypic data from the present study confirm that PSD resistance in MO/PSD-0259 is conditioned by a single dominant gene linked to Sat_317 and Sat_120 on MLG F (Rpsd1) (Figure 3).
Inheritance studies on PSD resistance in PI 80837 indicated that a single dominant gene conditioned resistance, which was different from that in MO/PSD-0259 (Jackson et al. 2005). Data from the current study have confirmed that the PSD resistance in PI 80837 is conditioned by a single dominant gene linked to Sat_177 and Sat_342 on MLG B2 (Rpsd2) (Figure 3), which is independent of MLG F.
In the previous study by Berger and Minor (1999), RFLP Marker A708_1 accounted for 62.2% of the phenotypic variation in one cross and 21% of the variation in another cross. Based on results from the integration of previous genetic linkage maps (IGLM), RFLP marker A708_1 was mapped 8.2 cM from Sat_120 and 11.2 cM from Sat_317 (Song et al. 2004). Our linkage map of Rspd1 (Figure 3), in reference to the current IGLM, would indicate that A708_1 could be approximately 16.8 cM from Rpsd1. This genetic distance might explain the lack of phenotypic variation accounted for by A708_1 in the F2 mapping populations used by Berger and Minor (1999). Several factors may affect genetic distances between loci. For example, the type of markers used for mapping, the specific pedigree of the population, and the population size. In our (AP 350 × MO/PSD-0259) F2 mapping population, we found that Sat_317 was 6.8 cM from Sat_120 (Figure 3). In our (AP 350 × PI 80837) F2 mapping population, we mapped Sat_177 11.5 cM from Sat_342, whereas the current IGLM separates these 2 markers by 12.5 cM in the same orientation (Song et al. 2004).
Many disease resistance genes in soybean have been found in closely linked clusters, particularly on MLG F (Ashfield et al. 1998). In our study, we used RFLP A708_1 to select SSR markers and mapped Rpsd1 to a chromosomal region on MLG F. This region appears to contain a cluster of resistance genes including Rps3 to Phytophthora sojae (Gordon et al. 2007, Figure 3), Rpv1 to Peanut mottle virus, Rsv1 to Soybean mosaic virus (Gore 2000; Koning et al. 2002) and Rpg1to bacterial blight caused by Pseudomonas syringae pv. glycinea (Shoemaker and Olson, 1993, Figure 3). Additionally, in our study, we mapped Rpsd2 on MLG B2 where resistance genes for Soybean mosaic virus (Rsv3) and P. sojae (Rps5 and Rps8) have been previously mapped (Diers et al. 1992; Jeong et al. 2002; Burnham et al. 2003). Overall, these findings indicated that Rpsd1 and Rpsd2 could be a part of closely linked resistance gene clusters as described by Ashfield et al. (1998) and Polzin et al. (1994).
Results of this research have identified and provided genetic markers linked to 2 different sources of PSD resistance. The use of MAS could facilitate the incorporation of these resistance genes into breeding lines and cultivars. MAS could also enable pyramiding of Rpsd1 and Rpsd2 into a single background, providing more durable resistance to PSD. Results from this study have shown that markers linked to Rpsd1 and Rpsd2 are not polymorphic between the 2 PSD-resistant parent sources, impeding the pyramiding process using MAS with currently available markers. This is probably due to the large genetic distance from each resistance gene to the respective markers and suggesting, as stated by Iqbal et al. (2001), that further SSR and/or other PCR-based marker development within gaps in the map is needed. With the development of new markers saturating these regions, impediments associated with the lack of polymorphisms between genotypes of different backgrounds would enable both MSA and gene pyramiding.
Funding
Arkansas Soybean Promotion Board and the Arkansas Agricultural Experiment Station.
The authors thank Dr Ken Korth, Dr Yinong Yang, Burl Seversike, Pamela Miller, Sherrie Smith, and Nathan Reyna for their help. We also thank the staff of the University of Arkansas Vegetable Substation at Kibler, AR, under the direction of Dennis Motes for field management. We also thank Dr H.C. Minor, University of Missouri for seed of MO/PSD-0259, and Keith Biludew from Agripro Seeds, Inc. Ames, IA, for seed of Agripro 350
References
Author notes
Corresponding Editor: Reid G. Palmer
