The Journal of Heredity 2001:92(3)
© 2001 The American Genetic Association 92:267-270
Molecular Mapping of Rxp Conditioning Reaction to Bacterial Pustule in Soybean
From the Department of Crop and Soil Sciences, University of Georgia, Athens, GA 30602-7272 (Narvel, Jakkula, Wang, and Boerma), Department of Plant Pathology, Georgia Experiment Station, Griffin, Georgia (Phillips), and School of Plant Science, Seoul National University, Suwon, Korea (Lee).
Address correspondence to H. R. Boerma at the address above or e-mail: rboerma;caarches.uga.edu.
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Abstract |
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The Rxp locus in soybean [Glycine max (L.) Merr.] that conditions reaction to bacterial pustule was mapped by simple sequence repeat (SSR) marker analysis. A population of 116 F4-derived lines from a cross between the resistant parent Young and the susceptible parent PI 416937 was used for mapping. The Rxp locus was mapped 3.9 cM from Satt372 and 12.4 cM from Satt014 on linkage group D2. Linkage associations were confirmed by identifying a close association between the SSR genotype at each locus identified as flanking Rxp and the bacterial pustule reaction of individual lines derived from a population different from the one used for mapping. A molecular pedigree analysis showed that bacterial pustule-resistant cultivars inherited the resistance gene rxp from the ancestral cultivar CNS based on their consistent genotypic pattern at flanking marker loci. Based on the results of the study, marker-assisted selection for rxp would be very effective.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Bacterial pustule (BP), caused by Xanthomonas campestris pv. glycines, is a foliar disease of soybean that is most common in the southern United States where high temperatures and humidity accompanied by sporadic heavy rainfall favor disease development (Bernard and Weiss 1973; Kennedy and Tachibana 1973). Symptoms of BP typically occur on leaves and include small, pale green spots with elevated pustules, which may develop into large necrotic lesions causing premature defoliation (Kennedy and Tachibana 1973). The effect of BP on the growth and development of soybean largely depends on infection levels, but only minor yield losses (4–11%) in the United States have been reported (Hartwig and Johnson 1953; Weber et al. 1966). Laviolette et al. (1970) evaluated resistant and susceptible lines in Indiana and reported no significant difference in yield among lines under high levels of artificial infestation.
Among the ancestors of domesticated North American soybean cultivars, field resistance to BP has only been identified in CNS which was found to be nearly immune to the disease (Feaster 1951; Hartwig and Lehman 1951). Hartwig and Lehman (1951) determined that BP resistance in CNS was conditioned by a single recessive gene, which latter was designated rxp (Bernard and Weiss 1973). Hwang and Kim (1987) reported that soybean cultivars with resistance derived from CNS exhibited no symptoms after separate inoculations with 20 different isolates of X. campestris pv. glycines. The rxp gene also conditions resistance to wildfire, caused by the bacterium Pseudomonas tabaci, a disease often associated with BP because of the pathogen's propensity to utilize BP lesions as an infection court (Kennedy and Tachibana 1973).
To identify linkage relationships with the Rxp locus, Palmer et al. (1992) evaluated a population segregating for isozymes and BP resistance. They found that Rxp was linked to the malate dehydrogenase (Mdh) locus with a recombination frequency of approximately 16%. This association defined linkage group (LG) 20 of the classical soybean genetic map, which consisted of isozyme, morphological, pigmentation, and pest-resistant loci.
With the advent of DNA marker technology, the soybean genetic map has been greatly expanded. A public soybean map was developed from three separate populations to form a consensus set of 20 homologous linkage groups, which presumably correspond to the haploid chromosome number in Glycine max (Cregan et al. 1999). The map contains 1423 unique marker loci and spans approximately 3000 cM. This highly saturated genetic map has greatly facilitated mapping loci for qualitative and quantitative traits.
Several of the classical marker loci were included on the public soybean map, but the exact location of the Rxp locus has not been determined. The general location of Rxp can be inferred from its linkage with Mdh. Mdh mapped on LG D2 in one population (Clark x Harosoy) and on LG H in another population (A81-356022 x PI 468916). It is highly suspected that Mdh is on LG D2 because Clark and Harosoy were reported to be polymorphic for Mdh (Shoemaker and Specht 1995) and possessed the same isozyme variants as those detected by Palmer et al. (1992). The Rxp locus could not be directly mapped because Clark and Harosoy were both susceptible to BP; therefore the map orientation of Rxp has not been resolved. This would limit the capacity of marker-assisted selection for BP resistance. The objectives of this study were to determine the precise map location of the Rxp locus and to confirm linkage associations in a population different from the one used for mapping. A pedigree analysis was conducted to determine if molecular markers could identify rxp in North American soybean cultivars with BP resistance derived from CNS.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Plant Materials
Soybean genotypes Coker 237, Young, PI 416937, and PI 97100 were used as parents to develop two populations that were segregating for BP resistance. Coker 237 and Young were highly productive cultivars that had previously been identified as resistant to BP (rxprxp). PI 416937 and PI 97100 were germplasm accessions from China that had previously been characterized as susceptible to BP (RxpRxp). A population of 106 F2-derived lines was developed from the cross PI 97100 x Coker 237 (PC). A population of 116 F4-derived lines from the cross Young x PI 416937 (YP) was developed by single-seed descent with each line originating from a different F2 plant.
Phenotypic Evaluation
The YP population was grown at one location in Athens, Georgia, and at one location in Plains, Georgia, during 1996. Three entries of Young and one entry of PI 416937 were included in each test. The PC population was grown at two locations in Athens, Georgia, and at one location in Blackville, South Carolina, during 1996. One entry of each parent and one entry of the BP-resistant cultivar Stonewall were included in each test. The experiments were conducted using a randomized complete block design. Each entry was grown in multiple-row plots. For each population, BP was evaluated on a single replication at each location that showed the greatest infestation. This totaled two observations for the YP population and three observations for the PC population. BP was scored by a coded system. Lines that had no BP symptoms were scored as 1 and lines that exhibited symptoms on any number of plants were scored as 4. This scoring convention was employed for linkage analysis, as described below.
Molecular Analysis
The parents and lines were sampled for DNA extraction by bulking leaves from at least 10 plants from each genotype. DNA was extracted using a CTAB protocol (Keim et al. 1988). Simple sequence repeat (SSR) markers were used for mapping. SSRs were amplified in PCR by using primers reported by Cregan et al. (1999) with the exception of Satt014 that was derived from Narvel et al. (2000). For SSR detection, one flanking primer was labeled with a fluorescent dye. Primer labeling and the PCR protocols were carried out according to Diwan and Cregan (1997). SSRs were analyzed on a 4.8% polyacrylamide gel mounted on a PE/ABI (PE Applied Bio[chsystems, Foster City, CA) model 377 automated DNA sequencer. Data were collected with DNA Sequencing Collection software version 2.5 and were analyzed with GENESCAN Prism software version 2.1, followed by analysis with GENOTYPER software (PE/ABI) for allele size determination. Allele sizes were rounded to an integer using the local Southern algorithm.
Linkage Analysis
The YP population was used for linkage analysis. Based on the assumption that rxp was on LG D2 due to its linkage with Mdh in the Clark x Harosoy population (Cregan et al. 1999), SSR markers that were near Mdh were used for linkage analysis. The genetic linkage map was constructed using the Kosambi mapping function in Gmendel version 3.0 (Holloway and Knapp 1993). SSR markers were analyzed as codominant data. The BP phenotype was used for linkage analysis and was analyzed as a dominant marker. Lack of BP symptoms implied that the line was derived from a rxprxp F4 plant. The presence of BP symptoms implied that a line was derived from a RxpRxp or Rxprxp F4 plant. For grouping markers into linkage groups, a minimum LOD (logarithm of the odds) of 3.0 and a maximum distance of 38 cM were used.
The 106 F2-derived lines from the PC population were genotyped with SSR markers identified as flanking the Rxp locus to confirm linkage associations. A molecular pedigree analysis was conducted to determine if BP-resistant cultivars of the southern United States inherited rxp from CNS. This analysis was preceded by genotyping the main ancestors of the southern soybean gene pool for the flanking markers. BP-resistant cultivars were identified in GRIN (Germplasm Resources Information Network), a web server supported by the USDA/ARS that provides information on plants and other organisms. A total of 12 cultivars were analyzed that had CNS in their pedigree and that were classified for BP reaction. DNA was obtained from each cultivar or ancestor by bulking leaves from five plants and was extracted as previously described. The cultivars were analyzed twice for each SSR marker to ensure accurate allele size determination.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Natural infestations of X. campestris pv. glycines were present at all locations as evidenced by consistent BP symptom development on the susceptible parents. No symptoms were observed on the resistant parents at any location. Making multiple observations greatly reduced the possibility of inaccurately classifying rxprxp plants due to variable natural field infestations across locations. A line was considered susceptible to BP if symptoms were detected on any number of plants within a line at any particular location. It was not possible to determine if lines were derived from RxpRxp or Rxprxp plants, because it was difficult to detect symptoms on all plants within a susceptible line. This may have been the result of variable field infestations at any given location. To circumvent potential misclassifications, the BP reaction was scored as a dominant trait. This scoring convention led to the expected phenotypic ratio (lines with symptoms:lines with no symptoms) of 3:1 in the PC population and 9:7 in the YP population. There were 83 lines that had symptoms and 23 lines that showed no symptoms in the PC population. This ratio satisfactorily fit the expected ratio (79.5:26.5) based on a chi-squared goodness-of-fit test (
2 = 0.61, P > .1). There were 65 lines that had symptoms and 51 lines that showed no symptoms in the YP population, which satisfactorily fit the expected ratio (65.25:50.75; 2 = 0.002, P > .95). The YP population was used to map the Rxp locus using the BP phenotype of the lines as a dominant marker. The YP population had previously been mapped with several hundred DNA markers with LG D2 defined by 14 markers that spanned an estimated 190 cM (data not shown). The Rxp locus mapped on LG D2 to a region defined by SSR markers (Figure 1). The Rxp locus was 3.9 cM from Satt372 and 12.4 cM from Satt014. These results are consistent with the map position of Mdh on LG D2 in the Clark x Harosoy population (Cregan et al. 1999), which was previously found to be linked to Rxp (Palmer et al. 1992).
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To confirm the map location of Rxp, 106 F2-derived lines were genotyped from the PC population for Satt372 and Satt135; Satt014 was not available for analyzing this population. Satt372 correctly classified 95% (101/106) and Satt135 correctly classified 92% (97/106) of the lines for their corresponding BP phenotypes (Table 1). These results were consistent with the relative map distance between each SSR marker locus and Rxp with fewer recombinants detected for the Satt372 marker class than for Satt135.
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CNS was a parent of Lee, which was one of the most widely grown cultivars in the southern United States and one of the most utilized parents in breeding programs during the early stages of cultivar development (Hartwig 1973). The development of BP-resistant cultivars was a priority among public breeders in the South for several decades (Boerma HR, personal communication). Because of these events, CNS has been identified as the most prominent ancestor of southern cultivars. Gizlice et al. (1994) reported that 17 ancestors constituted the majority of the southern U.S. genetic base and estimated that CNS had an average coefficient of parentage of 24.7% in public cultivars that were released between 1947 and 1988. Delannay et al. (1983) reported that CNS was present in the pedigree in each of the 48 southern cultivars they evaluated. Based on these characteristics, it might be possible to track rxp in southern soybean germplasm if CNS possessed unique or rare alleles at one or both SSR loci flanking the Rxp locus.
To test this hypothesis, 13 ancestors of the southern U.S. gene pool, including CNS, were genotyped for Satt014 and Satt372. The 13 ancestors combined have contributed approximately 85% of the alleles in southern cultivars based on the data reported by Gizlice et al. (1994). For Satt014, three alleles were detected across the 13 ancestors and none of the ancestors had the same genotype as CNS (Table 2). Two different banding patterns were detected for Satt372. With the exception of S-100, all the ancestors had a major band within a 244–259 bp range and a minor band at 347 bp. The reverse was found for S-100, which had a minor 247 bp band and a major 347 bp band. The allele that cosegregated with BP resistance in both the YP and PC populations was 250 bp; therefore it was assumed that the 247 bp fragment detected in S-100 was the true allele at Satt372. The only ancestor that had the same genotype as CNS at Satt372 was Lincoln (Table 2). Although CNS and Lincoln are 50% similar for Satt014 and Satt372, it should not limit the use of one or a combination of both markers to detect rxp because Lincoln made a relatively minor contribution to southern cultivars.
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The ability to detect rxp through SSR marker analysis was tested by genotyping 12 elite southern cultivars that had CNS in their pedigree and were classified as BP resistant. The 12 cultivars had the same genotype as CNS for Satt014 and Satt372 (Table 2). In addition, the BP-resistant cultivars that were used as parents in this study, Young and Coker 237, both had the same genotype as CNS. These results strongly support the hypothesis that the cultivars inherited rxp from CNS. Based on these results, it might be possible to select southern U.S. breeding lines or cultivars with known CNS parentage that possess rxp, but otherwise have an unknown reaction to BP. The accuracy of this method will depend on the extent of linkage disequilibrium that has been maintained between the flanking marker loci and the Rxp locus over decades of breeding.
As previously stated rxp confers near immunity to BP under field conditions. Symptoms have been detected on greenhouse-grown plants of CNS under heavy innoculation levels (Chamberlin 1962). Sharma et al. (1993) identified the soybean line P-4-2 from India that was resistant to BP when evaluated against high innoculation levels in the greenhouse. Manjaya and Pawar (1999) evaluated the genetic mechanism of resistance in P-4-2 and reported that it was controlled by duplicate recessive gene action. They indicated P-4-2 possessed resistance genes different from rxp, but they did not conduct any genetic tests to support this observation. This could be resolved by mapping the resistance genes from P-4-2 to determine if they map to a location different from the Rxp locus.
Rxp is the only locus controlling disease resistance in soybean that has been mapped on LG D2. Disease resistance gene clusters have been identified in soybean (Kanazin et al. 1996). Polzin et al. (1994) identified a resistance gene cluster on LG J that contained Rps2 for phytopthora root and stem rot and Rmd for powdery mildew and the Rj2 locus that controls Bradyrhizobium japonicum-mediated nodulation. Mian et al. (1999) mapped the Rcs3 gene for resistance to frogeye leaf spot disease near the cluster on LG J. Several resistance genes have been mapped to a region on LG F including Rps3 for phytopthora root and stem rot (Diers et al. 1992), Rsv1 and Rpv1 for soybean mosaic virus and peanut mottle virus, respectively (Roane et al. 1983), and Rpg1 for bacterial blight (Ashfield et al. 1998). Because disease resistance genes have been detected in clusters, other resistance genes may be located near Rxp on LG D2.
One of the most useful applications of a linkage map is marker-assisted selection for traits that may be unwieldy to evaluate by conventional means, such as pest resistance. Because BP has only a minor impact on soybean productivity, marker-assisted selection for rxp will likely not be a priority among soybean breeders. With increased efforts in expanding the genetic base of soybean through the introgression of exotic germplasm, however, selection for rxp might be needed in the future. The incorporation of exotic germplasm could increase the frequency of the susceptible allele that could heighten BP incidence. Based on the results of this study, marker-assisted selection for the resistance gene rxp would be very effective.
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Footnotes |
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Corresponding Editor: Reid G. Palmer
Received June 19, 2000
Accepted November 11, 2000
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