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a Syngenta Seeds, Inc., 317-330th St., Stanton, MN 55018
b Monsanto, 634 East Lincoln Way, Ames, IA 50010
c Dep. of Crop Sciences, Univ. of Illinois, 1102 S. Goodwin Ave., Urbana, IL 61801
d College of Agricultural and Life Sciences, UW-Madison, 1630 Linden Dr., Madison, WI 53706
Corresponding author (mike.bachman{at}syngenta.com)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: AFLP, amplified fragment length polymorphism • BSR, brown stem rot • MLG, molecular linkage group • PCR, polymerase chain reaction • PI, plant introduction • QTL, quantitative trait locus/loci • RFLP, restriction fragment length polymorphism • SSR, simple sequence repeat
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Selection for BSR resistance is hampered by a high level of environmental variation in the field (Chamberlain and Bernard, 1968) and labor-intensive, time-consuming assays in the greenhouse (Sebastian and Nickell, 1985; Sebastian et al., 1985). However, the use of molecular markers linked to Rbs genes could accelerate selection and eliminate the effects of environmental variation. Molecular markers could also facilitate pyramiding of Rbs loci which could provide more temporally or geographically stable resistance to P. gregata in the future. Stable BSR resistance sources are desirable given the historical failure of many monogenic disease resistance mechanisms in plants and reports of physiological specialization in P. gregata (Gray, 1971; Willmot et al., 1989). There is also evidence that while single Rbs resistance alleles usually confer high-level resistance, they do not provide immunity from disease; there are reports of BSR symptom development on soybean lines containing Rbs resistance alleles (Bachman et al., 1997a; Hanson et al., 1988; Nelson et al., 1989).
Several different molecular marker systems have been used successfully in soybean, although these systems can be limited by expense, labor requirements, or a lack of repeatability if used for marker-assisted selection (Denny et al., 1996; Mudge et al., 1997). Restriction fragment length polymorphism (RFLP) markers were among the first molecular markers to be used in soybean. However, RFLP markers have several disadvantages for use in marker assisted selection, including relatively low levels of polymorphism (Keim et al., 1989, 1992) and time-consuming protocols (Mudge et al., 1997). SSR markers are DNA sequences consisting of short tandem repeats of two to five nucleotides (core sequences) flanked by conserved DNA sequences. These markers can vary in length, depending on the number of core sequences positioned in tandem arrangement. Sequence length polymorphism can be assayed by amplification of these regions with the conserved flanking sequences used as primer templates in the polymerase chain reaction (PCR) (Akkaya et al., 1992). SSR markers have been widely utilized in soybean because they have high levels of sequence polymorphism, codominance, repeatability, and they are rapid and relatively inexpensive to use (Maughan et al., 1995; Mudge et al., 1997). Because many of these markers have been mapped in the soybean genome (Akkaya et al., 1995; Cregan et al., 1994), genes of interest can be placed relative to known markers.
Molecular markers have been utilized in soybean to identify a cluster of genes on Molecular Linkage Group J (MLG) involved in plant responses to fungal pathogens and a bacterial symbiont. Polzin et al. (1994) used RFLP markers to identify a cluster of three loci on MLG J involved in disease resistance and symbiosis. Comprising this cluster was a gene for resistance to Phytophthora rot (Rps2), a gene for resistance to powdery mildew (Rmd), caused by Microshaera diffusa Che. & Pk. and a gene involved in nodulation (Rj2). Webb (1997), using RFLP markers, mapped BSR resistance gene, Rbs3, to the same region on MLG J. Lewers et al. (1999), using RFLP and amplified fragment length polymorphism (AFLP) markers on members of the same soybean population studied by Webb (1997), mapped a major quantitative trait locus (QTL) (likely Rbs3) and a minor QTL for BSR resistance to the same region. More recently, the Rcs3 locus, conferring resistance to frogeye leaf spot, was mapped by SSR markers to the same gene cluster (Mian et al., 1999).
The studies outlined above indicate that disease resistance genes can be successfully mapped in soybean by molecular marker systems. Given the operative and efficient SSR marker system, a reliable greenhouse assay for BSR resistance (Sebastian et al., 1983), and populations segregating for Rbs1 and Rbs2 BSR-resistance loci, our objectives were to (i) identify SSR markers linked to Rbs1 and Rbs2 to facilitate marker-assisted selection; and (ii) map Rbs1 and Rbs2 in the soybean genome.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Rbs1 F2:3 Families
In 1984, a cross was made between L78-4094, a BSR resistant germplasm line carrying Rbs1, and susceptible cultivar Century (Wilcox et al., 1980). The F1 plants were grown and harvested in 1985. The F2 seed was kept in cold storage. In 1997, a portion of the F2 seed from each of four F1 plants was planted at the Crop Sciences Research and Education Center, Urbana, IL. Single plants were harvested and threshed individually to obtain F2:3 families.
Rbs2 F2:3 Families
In 1983, a cross was made between PI 437833, a BSR resistant accession carrying Rbs2, and susceptible cultivar Century. The F1 plants were grown and harvested in 1984. The F2 seed was kept in cold storage. In 1997, a portion of the F2 seed from one F1 plant was planted at Urbana. Single plants were harvested and threshed individually to obtain F2:3 families. In the winter of 1996, a cross was made between PI 437833, a BSR resistant accession carrying Rbs2, and susceptible cultivar Century 84 (Walker et al., 1986) in the greenhouse. The F1 plants were grown in the summer of 1996 to produce F2 seed. In 1998, a portion of the F2 seed from one F1 plant was planted at Urbana. Single plants were harvested and threshed individually to obtain F2:3 families. All crosses were verified in subsequent generations using phenotypic markers, including flower color, pubescence color, and hilum color.
Rbs2 Near-isogenic Lines
The Rbs2 near-isogenic lines used in this study were developed at the University of Illinois as described in Bachman et. al. (1997b). Briefly, a cross was made between BSR-susceptible cultivar Century 84 and PI 437833 (Rbs2). Segregating progeny were screened for BSR resistance and Rbs2/rbs2 heterozygotes were selected during inbreeding. Following four cycles of screening and selection for Rbs2/rbs2 heterozygotes, homozygous resistant (Rbs2/Rbs2) and susceptible (rbs2/rbs2) individuals were identified through progeny testing, and increased to generate F6 derived Rbs2 near-isogenic lines. The resulting five pairs of Rbs2 near-isogenic lines, each pair initially derived from a different F2 plant, were then increased for three generations and selected for uniform phenotype.
Inoculation with Phialophora gregata and Plant Culture
Soybean populations and lines were evaluated for BSR reaction after inoculation with a monoconidial isolate of P. gregata, designated PgOh2. This isolate was initially cultured from soybean tissue from Ohio (personal communication, 1994, L.E. Gray), and it was obtained from Dr. L.E. Gray, University of Illinois, USDA-ARS. (The isolate PgOh2 is currently maintained by Dr. Brian Diers, University of Illinois.) This isolate was chosen for this study on the basis of the following three criteria: (i) its ability to induce BSR foliar symptoms on susceptible soybean varieties, (ii) its relative avirulence against BSR resistance genes Rbs1, and Rbs2, and (iii) its stability over time in continuous culture (Bachman et al., 1997a; Bachman and Nickell, 1998; Bachman and Nickell, 2000a,b).
Inoculum of P. gregata was prepared as described by Bachman and Nickell (2000a). Briefly, cultures were initiated by transferring three agar plugs (each approximately 2.7 mm3) containing hyphal tips of an active, 30- to 40-d culture from soybean stem agar minimal medium (Allington and Chamberlain, 1948) to 100 mL soybean seed broth (100 g of soybean seed/L water steamed, strained, and autoclaved). Both stem agar and seed broth were made from susceptible cultivar Century 84. Stationary liquid cultures were incubated at 24°C in the dark. After 4 wk, seed broth cultures of P. gregata were ground for 75 s in a blender at high speed. The concentration of propagative fragments (mycelial fragments and conidia) was determined with a hemocytometer. Blended cultures were then diluted with distilled water to a concentration of 1.2 x 106 propagules/mL. Carboxymethyl cellulose was added to the suspension at a rate of 7.5 g/L to act as a sticking agent.
Inoculation of plants for greenhouse evaluation was conducted by a root-dip technique developed by Sebastian et al. (1983) and modified in accordance with the following methods. Seed was germinated in commercial grade sand in 10-cm-diam plastic pots and grown to the V1 growth stage (Fehr et al., 1971) at temperatures ranging from 18 to 24°C. Sand was rinsed from the roots of the seedlings. Five healthy-appearing seedlings were selected, and the roots were blotted dry with paper towels and dipped into a beaker containing 50 mL of P. gregata inoculum for 2 to 3 s. The seedlings were removed from the inoculum and placed into a 6- to 8-cm depression in steam-treated 1:1 sand:topsoil mixture filling a 15-cm-diam steam-sterilized clay pot. The remaining inoculum was then poured over the roots of the seedlings. The mixture of 1:1 sand:topsoil was used to cover the roots of seedlings to a level 1 to 2 cm below the cotyledons. Pots were placed on trough-shaped benches lined with 25 to 50 mm of commercial grade sand. Plants were maintained under a 14-h photoperiod at an average nighttime temperature of 18°C and an average daytime temperature of 24°C. All pots received approximately 300 mL of water daily. Pots were fertilized weekly with 150 mL of a nutrient solution containing 0.121 g N, 0.112 g P, 0.107 g K, 0.000044 g B, 0.00022 g chelated Fe, and 0.00011 g chelated Cu, Mn, and Zn.
Experimental Design for BSR Evaluation
Seventy-three F2:3 families segregating for Rbs1 and 77 F2:3 families segregating for Rbs2 (including 30 families derived from the cross of PI 437833 x Century and 47 families derived from the cross of PI 437833 x Century 84) were screened for BSR reaction in the greenhouse in the winters of 1997-1998 and 1998-1999 by the technique described above. The resistant and susceptible parents of each population, as well as each family segregating for Rbs1 and Rbs2, were represented by four pots (totaling approximately 20 plants). Families and parental controls for each population were arranged in a completely randomized design.
BSR Evaluation
Brown stem rot reactions of Rbs2 near-isogenic lines were confirmed in field tests in 1994 and 1995 (Bachman et al., 1997b), and in a greenhouse evaluation in the winter of 1996. Resistant and susceptible near-isogenic lines were easily distinguished by the absence and presence, respectively, of foliar BSR symptoms to or above the node bearing the first trifoliolate leaf (Hanson et al., 1988) (data not included).
Brown stem rot reaction to inoculation is influenced by the inoculation efficiency, genetic backgrounds of the material under evaluation, and the environment. As a result, BSR reaction data can be quantitative in nature, and phenotypic classification of segregating populations can be difficult. In this study, restricted population size necessitated the use of a reliable method of phenotypic classification of genetic families for BSR reaction. As a means of verifying the results of our phenotypic classification, genetic families segregating for Rbs1 and Rbs2 were phenotypically classified by two methods.
To utilize the first method of phenotypic classification, families were initially evaluated for BSR mean foliar severity. Mean foliar severity of BSR was calculated by rating each plant for height of foliar symptom progression (the proportion of total nodes with an expanded leaf showing BSR foliar symptoms) and averaging these values over all plants in a family. Brown stem rot mean foliar severity ratings were expressed as a decimal value from 0 to 1. Mean foliar severity values were arbitrarily divided into phenotypic classes with low (homozygous resistant), intermediate (segregating), and high (homozygous susceptible) means (Hanson et al., 1988; Willmot and Nickell, 1989) (data not included).
The second classification method for families was based on the ratio of resistant to susceptible plants within a family. To utilize this method, individual F3 plants within a family were phenotypically classified. Plants were classified as resistant if they had no disease or developed BSR foliar symptoms to a level below the first trifoliolate node, and susceptible if they developed BSR foliar symptoms to or above the first trifoliolate node (Hanson et al., 1988). Observed numbers of resistant and susceptible plants within a family were then compared, by Chi-square analysis, to expected numbers for segregation of a single dominant gene (by ratios of 1 resistant: 0 susceptible, 3 resistant: 1 susceptible, and 0 resistant: 1 susceptible)(data not included). A family was classified as resistant when the highest Chi-square probability was obtained from comparison to an expected ratio of 1 resistant: 0 susceptible. Likewise, a family was classified as segregating when the highest Chi-square probability was obtained from comparison to an expected ratio of 3 resistant: 1 susceptible. Finally, a family was classified as susceptible when the highest Chi-square probability was obtained from comparison to an expected ratio of 0 resistant: 1 susceptible. In the infrequent event of disagreement between the two classification methods, the family of interest was classified by the "Chi-square probability" method, because Chi-square analysis has been used in previous genetics studies involving BSR resistance (Hanson et al., 1988; Willmot and Nickell, 1989).
Tissue Sampling
Two weeks after inoculation (V2 growth stage), plants were tissue sampled. Approximately equal proportions of leaf tissue (young expanding trifoliolates) were harvested from 10 to 12 individual F3 plants within each family segregating for Rbs1 and Rbs2. This tissue was bulked to reconstruct the F2 plant from which those families were derived. Young trifoliolate leaves were collected from approximately 10 individual plants of each of the Rbs2 near-isogenic lines and bulked. Leaf tissue collected from families and near-isogenic lines was placed in plastic heat-seal bags, and immediately placed on ice. Tissue samples were frozen in liquid nitrogen, lyophilized for approximately 48 h, sealed in bags, and stored at -20°C.
DNA Extraction
DNA was extracted by the method of Saghai-Maroof et al. (1984). Approximately 0.75 g of freeze-dried soybean leaf tissue was powdered with a modified paint shaker. Following addition of 10 mL extraction buffer [50 mM tris, pH 8.0, 0.7 M NaCl, 10 mM EDTA, 1% (w/v) hexadecyltrimethylammonium bromide, 0.1% (v/v) 2-mercaptoethanol], the slurry was incubated for 60 min at 60°C with occasional swirling. After incubation, 10 mL of chloroform-octanol (24:1, v/v) was added, and the solution was mixed by inversion and centrifuged at 5125 x g for 10 min at 4°C. The aqueous phase was removed and the DNA was precipitated by adding 2/3 volume of isopropanol. Precipitated DNA was spooled onto a glass rod and washed in 20 mL of a 76% (v/v) ethanol/ 10 mM ammonium acetate solution. The DNA was then dissolved in 1.5 mL of 10 mM ammonium acetate/0.25 mM EDTA.
SSR Amplification and Viewing
All SSR primers used in this study were described by Cregan et al. (1999) and are listed at http://129.186.26.94/SSR.html; verified November 17, 2000. The Rbs2 near-isogenic lines were assayed for polymorphism with 154 SSR markers. Parents of progeny segregating for Rbs1 and Rbs2 were assayed for polymorphism with a set of 112 SSR markers from 20 linkage groups. Markers residing on Soybean Molecular Linkage Group J (Cregan et al., 1999) were tested initially because a cluster of disease resistance genes has been identified on that linkage group (Polzin et al., 1994; Webb, 1997). Subsequent analyses in parents of both Rbs1 and Rbs2 populations included SSR loci spanning the soybean genome. Polymorphic markers were then assayed on segregating progeny. Amplification of SSR loci was carried out as described by Akkaya et al. (1995). Polymerase chain reaction mixtures included 30 ng of genomic DNA, 1.5 mM Mg2+, 0.15 mM of 3' and 5' end primers, 200 mM each of dATP, dTTP, dCTP, and dGTP, 1x PCR buffer containing 50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% (v/v) Triton X-100, and 1 unit Taq polymerase in a total volume of 10 mL. Thirty thermal cycles were run, each included a 25-s denaturation step at 94°C, a 25-s annealing step at 47°C, and a 25-s extension step at 68°C. PCR products were separated with 3.0% (w/v) Metaphor (FMC BioProducts, Rockland, ME) agarose gels, stained with ethidium bromide, and visualized under UV light.
Statistical Analyses
Broad sense heritability estimates for BSR mean foliar severity were calculated for each population under study. The variance among families in a population was used as an estimate of total phenotypic variance. The variance among four replicates of each of two homozygous parental checks was pooled and used as an estimate of error variance for each population. Total genetic variance was calculated as the difference between total phenotypic variance and error variance. The broad sense heritability was estimated as the proportion of total phenotypic variance attributed to the total genetic variance.
The observed BSR reaction phenotypes and SSR markers were tested for goodness-of-fit to expected ratios for segregation of a single gene using Chi-square analysis.
Simple sequence repeat markers were analyzed using single factor analyses in SAS (SAS Institute, 1985) to determine the proportion of variation in BSR reaction that was explained by individual markers. Markers significant at P = 0.01 probability level were included in linkage analysis using the Kosambi function of Mapmaker/EXP 3.0 (Lander et al., 1987; Lincoln et al., 1992a). The BSR phenotypic reaction was coded as a marker (resistant, heterozygous, or susceptible) and mapped as a qualitatively inherited trait with respect to SSR markers. In an additional analysis, the BSR mean foliar severity value of Rbs1 and Rbs2 F2:3 families was treated as a quantitative trait, and putative quantitative trait loci (QTL) were identified and mapped by Mapmaker QTL 1.1 (Paterson et al., 1988; Lincoln et al., 1992b). Maps generated from Rbs1, Rbs2, and linked SSR markers were compared with a recently published molecular linkage map of soybean (Cregan et al., 1999) containing identical SSR markers.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The two methods used to classify BSR phenotypic reactions were in agreement for 71 of 73 families segregating for Rbs1 and 73 of 77 families segregating for Rbs2. Agreement between these methods provided evidence that either classification scheme could distinguish phenotypic classes within the pool of segregating families.
Results of Chi-square analyses indicated that SSR markers and BSR phenotypic reactions for families in both the Rbs1 and Rbs2 populations fit expected ratios for segregation of a single gene (Table 1).
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Marker Assisted Selection Using SSR Markers
On the basis of the data collected in this study, efficiency of selection for BSR reaction phenotype in populations segregating for the Rbs1 allele would be highest if Satt431alone were used. The marker Satt431 predicted the BSR reaction phenotype in 88% of the F2:3 families, whereas Satt215 predicted the correct reaction phenotype in only 62% of the families. Prediction of BSR reaction phenotype using flanking markers was less effective. When both Satt215 and Satt431 carried the marker alleles associated with a given BSR reaction, these marker loci predicted only 6% of the BSR reaction phenotypes of families. These trends in selection efficiency would be expected on the basis of the relatively large map distances between the SSR markers and Rbs1.
Selection efficiency for Rbs2-conferred resistance was not as high as that for resistance conferred by Rbs1. When used alone, SSR markers Satt244 and Satt431 predicted the BSR reaction phenotype in 82 and 70%, respectively, of the families segregating for Rbs2. Although marker assisted selection in this study was most efficient using Satt244, identification of markers with tighter linkage to Rbs2 would be desirable to improve efficiency of selection.
Genes conferring resistance to a single pathogen or to taxonomically diverse pathogens have been mapped to discrete clusters in maize, Zea mays L., (Bennetzen et al., 1991; Hulbert and Bennetzen, 1991), rice, Oryza sativa L., (Kinoshita, 1993; Mackill and Bonman, 1992; Yoshimura et al., 1983), tomato, Lycopersicon esculentum Mill., (Dickinson et al., 1993), barley, Hordeum vulgare L., (Wise and Ellingboe, 1985), flax, Linum usitatissimum L., (Islam et al., 1989; Mayo and Shepherd, 1980; Shepherd and Mayo, 1972), lettuce, Lactuca sativa L, (Kesseli et al., 1990, 1992, 1993; Maisonneuve et al., 1994), and soybean (Diers et al., 1992; Polzin et al., 1994; Yu et al., 1994). Related structure and/or function among genes in these clusters has provided evidence that resistance genes occur in multigene families and have a common origin (Parniske et al., 1997; Ronald, 1998; Song et al., 1997).
In soybean, a cluster of disease resistance genes including Rps2, Rmd (Polzin et al., 1994), Rcs3 (Mian et al., 1999), Rbs1, Rbs2 (present study), and Rbs3 (Webb, 1997; Lewers et al., 1999) has been mapped to Molecular Linkage Group J (Fig. 3). The loci Rps2 and Rmd are involved in resistance responses to Phytophthora sojae M.J. Kaufmann & J.W. Gerdemann and Microspaera diffusa Cooke & Peck, respectively (Buzzell and Haas, 1978; Kilen et al., 1974; Lohnes and Bernard, 1992). The Rcs3 gene conditions resistance to frogeye leaf spot in soybean, caused by Cercospora sojina K. Hara (Phillips and Boerma, 1982). The genes, Rbs1, Rbs2, and Rbs3 provide resistance to brown stem rot of soybean (Hanson et al., 1988; Willmot and Nickell, 1989). The BSR resistance gene, Rbs3, was mapped to this region of Molecular Linkage Group J using RFLP markers (Webb, 1997). In addition, Lewers et al. (1999) identified RFLP and AFLP markers in this region linked to two QTL associated with BSR resistance. Because the populations under study by Webb (1997) and Lewers et al. (1999) were the same, it is likely that the major QTL found in the latter study was Rbs3. An important finding in the study by Lewers et al. (1999) was the association of a minor QTL for BSR resistance with a resistance gene analog. Multiple resistance gene analogs have been mapped to this region of Molecular Linkage Group J (Kanazin et al., 1996), and the possibility exists that these resistance gene analogues correspond to one or more of the BSR resistance genes identified in this region, or to unique BSR resistance loci.
Polzin et al. (1994) also mapped another locus, Rj2, to the gene cluster discussed above (Fig. 3). Alleles at this locus have been implicated in nodulation response to specific strains of Bradyrhizobium japonicum (Caldwell, 1966). This region on MLG J is therefore involved in both defense responses to diverse fungal pathogens and symbiotic relationships with a bacterium.
In the initial search for SSR loci polymorphic between BSR resistant and susceptible parents, Molecular Linkage Group J was targeted because it contained a cluster of genes for resistance to several soybean pathogens. This approach proved successful in locating BSR resistance loci, Rbs1 and Rbs2, and the Rcs3 locus conferring resistance to frogeye leaf spot (Mian et al., 1999). The presence of multiple resistance gene analogs in this region provides evidence for the existence of additional genes conferring resistance to soybean pathogens, and this region may be the focus of future attempts to map or clone resistance loci.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication May 11, 1999.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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