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CELL BIOLOGY & MOLECULAR GENETICS

Molecular Mapping of the Rcs3 Gene for Resistance to Frogeye Leaf Spot in Soybean

^a Dep. of Crop and Soil Sciences, Univ. of Georgia, Athens, GA 30602-7272 USA
^b Dep. of Plant Pathology, Georgia Exp. Stn., Griffin, GA 30223 USA

rboerma{at}uga.edu

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
Frogeye leaf spot (FLS) (caused by Cercospora sojina Hara) is a foliar disease of soybean [Glycine max (L.) Merr.] that causes significant yield loss in southeastern USA. The Rcs 3 gene in soybean has been reported to condition resistance to all known races of C. sojina. Molecular mapping of the Rcs3 gene will be helpful in breeding soybean for resistance to FLS. The objective of this study was to map the Rcs₃ gene with DNA markers. The parents, 123 F₂ plants, and the F_2:3 families from the cross of `Blackhawk' (susceptible) x `Davis' (resistant and original source of the Rcs3 gene) were scored for FLS reaction in the greenhouse after inoculation with race 5 of C. sojina. In addition, `Wright' and the Wright⁶-Rcs 3 (near isoline of Wright) were also scored for FLS by the same protocol. A resistant and a susceptible DNA bulk were created by pooling the DNA of 15 resistant and 15 susceptible F₂ plants, respectively, from the Blackhawk x Davis population. The two bulks and resistant–susceptible near isolines (NILs) of Wright were screened with simple sequence repeat (SSR) markers from soybean linkage groups with known disease resistance gene clusters. The bulks and the NILs of Wright both showed a putative association between the Rcs 3 locus and a SSR marker Satt244 from linkage group (LG) J. A molecular map of the relevant segment of the LG J for the Blackhawk x Davis population was then created by screening the DNA of 95 F₂ plants with the available polymorphic SSR and RFLP markers. We have mapped the Rcs3 gene for resistance to FLS in soybean near a previously known disease resistance gene cluster on LG J. The gene was closely linked to SSR markers Satt244 and Satt547. Close proximity of this gene with these SSR markers provides soybean breeders with an opportunity to use these markers for marker assisted selection (MAS) for FLS resistance in soybean.

Abbreviations: FLS, frogeye leaf spot • LG, linkage group • MAS, marker assisted selection • NILs, near isolines • RFLP, restriction fragment length polymorphisms • SSR, simple sequence repeats

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
FROGEYE LEAF SPOT

is primarily a foliar disease of soybean even though seeds, pods, and stems can also be infected. The incidence of FLS in soybean is dependent on the growing conditions (Akem and Dashiell, 1994), and the disease is favored by warm humid environments (Sinclair and Backman, 1989). The yield loss from FLS is mainly the result of reduced photosynthetic area and premature defoliation (Akem and Dashiell, 1994). Frogeye leaf spot causes significant yield losses in warm-humid environments of southeastern USA. Mian et al. (1998) reported a yield loss of up to 31% for four susceptible soybean cultivars compared with the respective resistant NILs in the southern USA. Comparisons of FLS resistant and susceptible soybean breeding lines in the USDA Uniform Tests indicated yield losses varying from 10% at Quincy, FL (Hartwig and Edwards, 1989) to 30% at Tallassee, AL (Hartwig, 1990). Greater than 60% yield loss due to FLS has been reported in the tropical environment of Nigeria (Dashiell and Akem, 1991).

At least five races of the fungus have been reported in the USA (Phillips and Boerma, 1981). Twenty-two races of C. sojina were identified in Brazil (Yorinori, 1992). In a recent study designed to further elucidate the extent of pathogen variability and to define a set of differential cultivars to aid in race identification of C. sojina, D.V. Phillips identified over 40 races of the fungus on a set of 38 differential cultivars (unpublished data, 1998). Three single genes conditioning resistance to C. sojina are currently recognized by the Soybean Genetics Committee. The gene Rcs1 in `Lincoln' was the first gene found that conferred resistance to race 1 of C. sojina (Athow and Probst, 1952). Rcs2 for resistance to race 2 was identified in `Kent' (Athow et al. 1962), and Rcs3 was identified in Davis conferring resistance to race 5 (Phillips and Boerma, 1982). The Rcs3 gene in Davis conferred resistance not only to race 5, but also to all other reported races in the USA (Phillips and Boerma, 1982), and all known Brazilian isolates (Yorinori, 1992).

Screening of a large number of soybean plants in early segregating generations for FLS requires greenhouse facilities and technical skills for C. sojina culture maintenance, preparation of inoculum, inoculation techniques, and scoring of the plants for the disease expression. Marker assisted selection for this trait could be an attractive alternative in that the need for C. sojina culture maintenance, inoculation, and retesting for FLS in later generations could be greatly reduced.

Identifying molecular markers linked to a particular disease resistance gene of interest can be laborious and time consuming. Two mapping strategies have been suggested to make the process efficient. These are bulked segregant analysis (Michelmore et al., 1991) and comparison of NILs for the trait (Muehlbauer et al., 1988). Bulked segregant analysis involves comparing two pooled DNA samples of individuals from a segregating population originating from a single cross. For each bulk, the individuals are identical for the targeted trait or gene (e.g., resistant vs susceptible to a particular disease), but are random for all other genes. Such DNA bulks are then analyzed to identify markers (polymorphic between the bulks) that are putatively linked to the targeted gene(s). Bulked segregant analysis has been used successfully by a number of investigators for identification of molecular markers linked to genes of interest (Giovannoni et al., 1991; Reiter et al., 1992; Barua et al., 1993; Williams et al., 1993).

The use of NILs is based on the concept that the DNAs of the recurrent parent and its NIL are mostly identical except in a small percentage of the genome. The genomes of NILs obviously differ in the region of the introgressed gene. Of the few polymorphic markers identified between NILs, some are linked to the introgressed gene of interest and the rest are scattered at random throughout the genome. Thus, comparing the DNA of the recurrent parent, donor parent, and the derived NILs can be efficient in identifying markers linked to a trait of interest. A number of researchers have successfully used this approach for identifying molecular markers linked to genes of interest (Paran et al., 1991; Diers et al., 1992; Van der Beck et al., 1992; Barua et al., 1993). However, there are advantages in using bulked segregant analysis over NILs. There is a minimal chance that regions unlinked to the target gene will be polymorphic between the bulked DNAs from many individuals. As bulked segregant analysis detects polymorphic loci using a segregating population, all loci detected will segregate and can be mapped (Michelmore et al., 1991). Creation of NILs is more time consuming than creation of a segregating population. Jean et al. (1998) found that the chance occurrence of shared homozygosity at specific unlinked chromosomal regions in the bulk limited the efficiency of bulked segregant analysis, while the efficiency of using NIL comparisons was limited by residual DNA from the donor genotype at scattered sites throughout the genome of the NILs. They also stated that the simultaneous screening of bulks and NILs can increase the chances of success in identifying markers linked to a gene of interest.

The objective of this research was to map the Rcs3 gene in soybean with molecular markers. Since disease resistance gene clusters in soybean have been reported by a number researchers (Yu et al., 1996; Kanazin et al., 1996; Polzin et al., 1994), our premise was that the SSR markers from soybean LGs harboring the disease resistance gene clusters have a higher probability of being linked to the Rcs3 locus than randomly selected SSR markers from the soybean molecular map (Cregan et al., 1999). Thus, we initiated our screening with the SSR markers from the genomic regions of soybean with known disease resistance gene clusters.

	Materials and methods

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
The Wright-Rcs3 NIL was created by backcrossing the Rcs3 gene from Davis into FLS susceptible cultivar Wright. The specific parentage was Wright⁶ x Davis. In each backcross generation, the F₁ plants were inoculated with the BG isolate of C. sojina. This isolate was previously shown to detect the Rcs3 gene for resistance to race 5 of C. sojina. In addition, a segregating population of 123 F₂ plants was created from a cross of FLS susceptible cultivar Blackhawk and resistant cultivar Davis.

Phenotypic Assay
The parents (Davis and Blackhawk) and 123 F₂ plants were evaluated in the greenhouse at the Georgia Experiment Station in Griffin, GA, during the summer of 1997. The plants were grown in 10-cm plastic pots on a greenhouse bench. At the V3 stage of development (Fehr and Caviness, 1977), the seedlings were inoculated with race 5 of C. sojina. Cultures of race 5 of C. sojina were maintained and the inoculum was produced on a medium composed of equal parts of soybean stem agar and lima bean agar (Difco). Conidial suspensions were made by flooding colonies of the fungus growing on agar in petri plates with sterile water and lightly scraping colonies to dislodge conidia. The suspensions were passed through several layers of cheesecloth to remove large mycelial fragments. Conidial suspensions from agar cultures were adjusted to a concentration of 6 x 10⁴ spores per milliliter. One trifoliolate leaf per plant was inoculated by atomizing a conidial suspension (2.5 mL) onto the upper and lower leaf surfaces. The inoculated leaves were enclosed in clear plastic bags for 48 hr to maintain high relative humidity. Disease ratings were made 14 d after inoculation. Since the FLS resistance in this population is known to be controlled by the single dominant Rcs3 locus, the FLS was scored as a qualitative trait (i.e., susceptible vs. resistant). Plants that showed numerous large spreading lesions were classified as susceptible, and each plant was given a score of 1 (to indicate that these plants were identical to the homozygous susceptible female parent, Blackhawk, in their FLS reaction). Plants that showed no lesions or only small lesions or flecks were classified as resistant, and each plant was given a score of 4 (to indicate that the FLS reactions of the heterozygous resistant plants were similar to that of the homozygous resistant plants and the paternal resistant parent Davis). The scores of 1 and 4 were assigned to the FLS susceptible and FLS resistant F₂ plants, respectively, so that these FLS scores could be mapped as a dominant gene on a molecular map of this soybean population. Plants classified as resistant were reinoculated to eliminate possible escapes. At maturity, seeds were harvested from the F₂ plants to create F₂-derived families.

To confirm the FLS reactions of the F₂ plants, the corresponding F₂-derived families were also evaluated for FLS reactions. The evaluation of F₂-derived families in the greenhouse was accomplished by the same protocol as described above for the screening of F₂ plants, except a minimum of 12 plants were evaluated for each family when available (16 families had less than 12 plants). The plants were grown in rows in trays rather than in plastic pots. Based on the FLS reaction of plants in each family, the initial FLS scores of the corresponding F₂ plants were confirmed. A F₂ plant producing a family with all susceptible plants was classified as susceptible, and was given a score of 1 as described under the F₂ plant scoring procedure. A F₂ plant producing a family with all resistant plants or a mixture of resistant and susceptible plants was classified as resistant, and was assigned a score of 4.

Genotypic Assay
For DNA extraction, young expanding leaves were harvested from the new growth on the same plants that were used for the phenotypic assay. The DNA extraction protocol was described earlier by Mian et al. (1996). Based on the F₂ FLS scores, the DNA from 15 randomly chosen FLS resistant F₂ individuals (from the 123 F₂ individuals screened in the greenhouse) were pooled to create the resistant bulk. Similarly, the susceptible bulk was created by pooling the DNA of 15 FLS susceptible individuals. Any marker that is polymorphic between these two DNA pools could be linked to the Rcs3 gene conferring resistance to FLS. The two parents of the F₂ population (Blackhawk and Davis), Wright, and Wright-Rcs3 NILs were also screened along with the bulks to find the markers linked to Rcs3. The NILs were included in our initial screening to support the result of bulked segregant analysis for the presence or absence of the linkage of a molecular marker with the Rcs3 gene. We selected the SSR markers from the USDA LGs E, F, G, J, and N that contain known disease resistance gene clusters for screening, rather than screening randomly chosen SSR markers.

SSR and RFLP Screening Protocols
The SSR primer sequences and aliquots of unlabeled SSR primers for this research were kindly provided by Dr. P.B. Cregan, USDA-ARS, Beltsville, MD. The sequence information for more than 600 soybean SSR primers including the ones used in this research are now publicly available from the USDA Internet site http://129.186.26.94/ssr.html; verified July 19, 1999). The SSR primer synthesis and fluorescent 5'-end labeling of the forward primer of each primer pair was done by PE-ABI (Foster City, CA) using phosphoramidite chemistry. The forward primers were labeled either with blue (6-Fam), green (Hex), or yellow (Ned) fluorescent tags. The PCR reactions were prepared according to the protocol of Diwan and Cregan (1997). The cycling consisted of 25 s of denaturation at 94°C, 25 s annealing at 46°C, and 25 s of extension at 68°C for 32 cycles on an ABI 9700 thermocycler. For each soybean line, the PCR products of a number of SSR markers (usually 3–5) with different fluorescent labels and/or with different allele sizes were pooled together. The sample of combined PCR products was loaded and separated on a ABI Prism 377 DNA sequencer (AB-PEC, Foster City, CA). GeneScan software (AB-PEC, Foster City, CA) was used for gel image analysis. The Genotyper software (AB-PEC, Foster City, CA) was used for accurate characterization of the alleles and for automated data output. In addition to the SSR markers, several RFLP markers were screened according to the protocol described by Mian et al. (1996).

Data Analysis
The linkage map was constructed with marker data and the FLS scores using Kosambi map function of Mapmaker/Exp (Lander et al., 1987). For combining markers into a linkage group, a minimum LOD of 3.0 and maximum distance of 50 cM were used.

	Results and discussion

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
At the time of scoring of FLS reaction in the greenhouse, Blackhawk showed typical susceptible reaction and Davis had the expected resistant reaction to FLS (Phillips and Boerma, 1981). By evaluating FLS reactions of the F₂-derived families, we confirmed the initial FLS scores of each F₂ plant. We identified seven F₂ plants that were misclassified in their initial scoring. On the basis of the confirmed FLS reactions, 29 of the 123 F₂ plants were susceptible and the remaining 94 plants were resistant. Chi-square analysis of the FLS scores showed a good fit to a 3 resistant:1 susceptible ratio indicating that the resistance was conditioned by a single gene with complete dominance. This is in agreement with our previous report that the dominant Rcs3 allele at the Rcs3 locus conditions resistance to race 5 of C. sojina in soybean (Phillips and Boerma, 1982).

Selection of SSR markers from the soybean LGs with known disease resistance gene clusters reduced the number of SSR markers to be screened by 80% compared with the number of markers needed for a random genome-wide search. This strategy of screening SSR markers proved to be an efficient and effective one. After the screening of 50 SSR markers, we identified an SSR marker, Satt244, that showed linkage with the Rcs3 gene (Fig. 1) .

View larger version (28K): [in this window] [in a new window]   Fig. 1 Gel image (from an ABI Prism 377 DNA sequencer using GeneScan analysis) for Wright, Wright-Rcs3 NIL, Blackhawk, Davis, FLS resistant bulk, and FLS susceptible bulk DNA for SSR markers Satt244 and Satt431. The NED labeled Satt244 is shown in yellow and the 6-FAM labeled Satt431 is shown in blue. The in-lane size standards are shown in red and the estimated number of base pairs (bp) for each in-lane size standard is shown on the left hand side of the gel

After finding the potential linkage of SSR marker Satt244 on LG J with the Rcs3 locus, 95 F₂ individuals were scored with four polymorphic SSR markers (Sct_001, Satt244, Satt431, and Satt547) and three polymorphic RFLP markers (A724_1, B032_1, and K375_1) chosen from the same genomic region where Satt244 is mapped on LG J (Cregan et al., 1999). Six (three SSRs and three RFLPs) of the seven polymorphic markers screened for the 95 F₂ plants from the Blackhawk x Davis cross linked together to form a segment of the soybean LG J (Fig. 2) . These six markers mapped in the same order as they were mapped previously (Cregan et al., 1999). Sct_001 initially remained unlinked (using a LOD min of 3.0 and max distance of 37 cM Kosambi). However, with a relaxed criteria for LOD min and max distance Sct_001 linked at 42 cM from RFLP marker K375_1 (Fig. 2).

View larger version (14K): [in this window] [in a new window]   Fig. 2 The section of linkage group (LG) J of the Blackhawk x Davis population showing the position of molecular markers (SSR and RFLP) and the Rcs3 gene for resistance to frogeye leaf spot (FLS) in soybean. A marker name and position are shown on the left and the estimated map distances (cM) are shown on the right of the LG. A Satt or a Sct_ prefix indicates a SSR marker locus. An RFLP marker locus is identified with a probe designation (a capital letter code with a number code) followed by a dashed number code

Polzin et al. (1994) reported a resistance gene cluster consisting of Rps2, Rmd, and Rj2 all located within a 3.8-cM region on soybean LG J. Rps2 conditions resistance to phytophthora root and stem rot (causal agent Phytophthora soja M.J. Kaufmann & J.W. Gerdemann), Rmd conditions resistance to powdery mildew (causal agent Microsphaera diffuse Cook & Peck), and Rj2 controls Bradyrhizobium japonicum mediated nodulation. Our results show that the Rcs3 locus is located near (within 14 cM) this gene cluster. In a recent report, Ashfield et al. (1998) have mapped Rpg1 gene for resistance to bacterial blight (causal agent Pseudomonas syringae pv. glycinea) in soybean to another known resistance gene cluster on soybean LG F. These findings support the idea that many of the unmapped resistance genes in soybean are probably located in the genomic regions with already known resistance gene clusters or in gene cluster(s) yet to be discovered. The use of SSR markers in the analysis precludes any ambiguity regarding duplicated regions in the soybean genome as could be the case with RFLP markers (Shoemaker et al., 1996).

As mentioned earlier, DNA of 95 of the 123 F₂ plants were used for mapping the Rcs3 locus. The DNA of the remaining 28 F₂ plants were screened with the two SSR markers (Satt244 and Satt547) closely linked to the Rcs3 locus. The genotypic scores for the two markers for each plant were then used to predict the FLS reaction of the corresponding F₂ plant. The data of the two markers, either individually or jointly, predicted the FLS reactions of all 28 plants correctly (Table 1) . These results support our conclusion regarding the close linkage of the two SSR markers to the Rcs3 locus. If only one SSR marker is to be used for selection, we recommend Satt244 instead of Satt547, because on the basis of our analysis Satt244 is more closely linked to the Rcs3 locus.

	ACKNOWLEDGMENTS

 
The authors thank Perry B. Cregan, USDA-ARS, Beltsville, MD, for providing the sequences and the aliquot of the soybean SSR primers. Dr. Cregan's technical advice regarding the use of the SSRs in this research is also appreciated.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION Materials and methods Results and discussion REFERENCES

 
This research was funded by state and Hatch funds allocated to the Georgia Agric. Exp. Stn. and by a grant from the Georgia Commodity Commission for Soybeans.

Received for publication April 13, 1999.

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