Mapping Genes Conferring Resistance to Phytophthora Root Rot of Soybean, Rps1a and Rps7 (original) (raw)

Abstract

A linkage map was constructed for two Phytophthora sojae Kauf. +Gerd. root rot resistance genes, Rps1a and Rps7, in soybean (Glycine max (L.) Merr.) using microsatellite or simple sequence repeat (SSR) markers. An F2 population consisting of 81 individuals derived from a cross between OX281, which carries Rps7, and Mukden, which carries Rps1a, was used as the mapping population. A linkage map consisting of 10 SSR markers was first constructed using the computer software MapMaker/EXP 3.0. Rps1a and Rps7 were then placed at two different loci in the same linkage group with LOD scores of 2.88 and 9.16, respectively. Rps1a and Rps7 were linked at a distance of 13.8 cM. Rps1a was flanked by Satt159 (0.7 cM) and Satt009 (3.2 cM). Rps7 was flanked by Satt009 (10.6 cM) and Satt125 (29.1 cM).

Phytophthora root rot, caused by Phytophthora sojae Kauf. +Gerd., is one of the most destructive fungal diseases of soybean (Glycine max (L.) Merr.) in North America. Seven different genetic loci for resistance have been reported (Anderson and Buzzell 1992; Athow and Laviolette 1982; Athow et al. 1980; Bernard et al. 1957; Buzzell and Anderson 1981; Kilen et al. 1974; Mueller et al. 1978). Fourteen genes, including six (Rps1a, Rps1b, Rps1c, Rps1d, Rps1e, and Rps1k) at locus Rps1, three (Rps3a, Rps3b, and Rps3c) at locus Rps3, and one at each of the other five loci Rps2, Rps4, Rps5, Rps6, and Rps7, are responsible for resistance to various physiological races of the pathogen (Schmitthenner 1999). The seven loci (Rps1, Rps2, Rps3, Rps4, Rps5, Rps6, and Rps7) have now been placed on the linkage map of soybean (Anderson and Buzzell 1992; Demirbas et al. 2001; Diers et al. 1992; Polzin et al. 1994). Rps1 and Rps7 have been located to the same linkage group (Anderson and Buzzell 1992).

By comparing previous studies on the mapping of Rps1 and Rps7, we found that discrepancies exist in the relative locations of Rps1, Rps7, and molecular markers on the linkage maps of separate studies using different mapping populations (Anderson and Buzzell 1992; Cregan et al. 1999; Diers et al. 1992; Lohnes and Schmitthenner 1997). Anderson and Buzzell (1992) reported a 14.6 cM distance between Rps1 and Rps7.Diers et al. (1992) reported a 6 cM linkage of Rps1a with RFLP marker A280-1, while Lohnes and Schmitthenner (1997) reported a 35.1 cM distance between Rps7 and A280-1. The distance between Rps1 and A280-1 would be 20.7 cM if we use results from the studies of Anderson and Buzzell (1992) and Lohnes and Schmitthenner (1997) to infer the location of Rps1. But the distance between Rps7 and A280-1 would be 14.7 cM if we use results from the studies of Anderson and Buzzell (1992) and Diers et al. (1992) to infer the location of Rps7. Any linkage between molecular markers and the Phytophthora resistance genes at these two loci based on these results could turn out to be inaccurate. If so, map-based cloning and marker-assisted selection using this linkage information could be misleading. A study using a strategy with Rps1, Rps7, and linked molecular markers segregating in one mapping population could provide convincing clarification for the linkage relationship. Unfortunately none of the previous studies used this strategy.

Previous studies on mapping Rps1 and Rps7 provide linkage relationships either only between Rps1 and Rps7, or only between Rps1 and molecular markers, or only between Rps7 and molecular markers. Anderson and Buzzell (1992) studied the segregation of Rps1 and Rps7 using classic methods and found that they were linked with a genetic distance of about 12.5 map units, or 14.6 cM, but no molecular markers were used to study the linkage. Diers et al. (1992) used restriction fragment length polymorphism (RFLP) markers to study the linkage between molecular markers and Rps1. They reported that markers pA-71 and pA280-1 are linked with Rps1, with genetic distances of 2 cM and 6 cM, respectively, but Rps7 was not included in the study and the linkage results seemed to be preliminary. Lohnes and Schmitthenner (1997) placed Rps7 on maps consisting of three microsatellite or simple sequence repeat (SSR) markers and two RFLP markers. However, Rps1 did not segregate in the mapping population. The linkage relationship between Rps1, Rps7, and molecular markers has relied on inference based on results of previous studies or integration of information from separate studies, which could be inaccurate. The linkage map that Lohnes and Schmitthenner (1997) constructed by integrating the information from their own study with that from the studies of Anderson and Buzzell (1992), Diers et al. (1992), and Shoemaker and Specht (1995) showed that the RFLP markers R022-1 and K395-2 were only 1.1 and 10.9 cM, respectively, from Rps7. The linkage information based on this integration may, however, be inaccurate.

In this study we developed a mapping population by crossing OX281 (rps1a, Rps7) and Mukden (Rps1a, rps7) so that Rps1, Rps7, and linked SSR markers segregated in the same population. A genetic linkage map, consisting of 10 linked SSR markers, was constructed for Rps1a and Rps7. SSR markers tightly linked with Rps1a and Rps7 were identified. These markers could be useful for map-based cloning of Rps1a and Rps7 genes and for marker-assisted selection in future soybean breeding programs involving Rps1a and Rps7 genes.

Materials and Methods

Plant and DNA Materials

An F2 population consisting of 81 individuals derived from the mating of parents OX281 and Mukden was used as the mapping population. OX281, a line derived from a cross of two Harosoy isolines, carries the resistance gene Rps7, but lacks the resistance gene Rps1a. Mukden (PI 50.523Q) carries Rps1a, but lacks Rps7 (Anderson and Buzzell 1992). Loci Rps1 and Rps7 were previously demonstrated to be linked (Anderson and Buzzell 1992). So Rps1a and Rps7 were assumed to be linked in repulsion phase in this mapping population. After the cross was made, F1 seeds were harvested and sown and F2 seeds were harvested for use of this study. Leaf samples were taken from the F2 individuals and DNA was isolated from the leaf samples according to the procedures described by Yu et al. (1999). For disease resistance tests, F3 progeny derived from individual F2 plants were used.

Disease Resistance Screening

Pathogen inoculation and criteria for determining disease resistance of seedlings followed the procedures described by Buzzell and Anderson (1992). For each of the 81 F2:3 lines, two sets of 12 seven-day-old seedlings were inoculated, one set with an isolate of race 1 (B77R1.55) and the other set with an isolate of race 35 (C21R12.29) of P. sojae (cultures obtained from Dr. Anne Dorrance, Ohio State University, OARDC, Wooster, OH 44691). Inoculated plants were incubated in humid conditions under plastic covers at 25°C for 48 h. Seedling reaction (alive versus dead) was recorded for each plant 3–5 days after inoculation. Plants without discoloration due to infection were considered escapes. Escapes and plants killed by the wounding operation but without infection were not counted. For those F2:3 lines with ambiguous dead/alive symptoms or with results conflicting with repulsion-phase linkage between Rps1a and Rps7 (which meant recombination occurred between loci Rps1a and Rps7), another 12 seedlings were tested to clarify dead/alive symptomatology or to confirm the recombination results. Twelve more seedlings were tested if further confirmation was necessary. The probability of misclassifying Rps/rps lines as rps/rps was 0.2512 = 6 × 10−6, and that of misclassifying Rps/rps lines as Rps/Rps was 0.7512 = 0.032 based on an assay of 12 plants per line.

PCR Primer Syntheses

Oligonucleotide sequences for PCR primers were mined from the SoyBase Internet site (http://129.186.26.94/SSR.html) constructed by the USDA-ARS Plant Genome Program, Cornell University, and Iowa State University. One oligonucleotide of each primer pair was tailed at its 5′ end with M13 universal forward primer sequence (19 bp). This tailed sequence for each primer pair was analyzed with computer software Gene Runner 3.05 (Hasting Software, Inc.) to avoid a possible high degree of hairpin structures within the oligonucleotide caused by tailing of M13 forward primer sequence, which would be unfavorable for PCR. If an unfavorable structure, whose annealing temperature was higher than 42°C, existed in the sequence, the other sequence of the primer pair was chosen for tailing. PCR primers were synthesized by either GENOSYS (Genosys Biotechnologies Inc., The Woodlands, TX) or the University of Guelph (Guelph, Ontario, Canada).

PCR Amplification and Detection

The LI-COR Global Edition IR2 System (LI-COR® Biotechnology Division, Lincoln, NE) (dual-laser system) was employed for PCR amplification and detection. PCR amplifications were performed in 96-well microtiter plates using the Perkin-Elmer GeneAmp PCR 9600 system. PCR reactions were in 10 μl mixes containing 25 ng genomic DNA, 1.5 mM Mg+2, 0.2 mM each dNTP, 1× PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 0.0001% gelatin), 0.05 μM tailed SSR primer pair, 0.05 μM M13 forward primer labeled with either IRD700 (with absorption maximum at 795 nm) or IRD800 (with absorption maximum at 685 nm) dye (LI-COR® Biotechnology Division, Lincoln, NE), and 0.4 units of DNA Taq polymerase (GIBCO BRL). The amplification temperature profile was as follows: 2 min at 94°C, followed by 35 cycles of 45 s at 94°C, 45 s at 47°C, and 45 s at 72°C, and then 5 min at 72°C. After PCR, two PCR mixes, one with M13 primer labeled with IRD700 dye and the other labeled with IRD800 dye, were mixed and detected together as one sample in order to increase efficiency. Four microliters of LI-COR stop buffer (47.5 ml deionized formamide, 20 ml 0.5M EDTA, 0.5 ml H2O, and 40 mg bromophenol blue) were added to each PCR mix and the PCR mixes were denatured for 5 min at 94°C and then put on ice for 5 min. The PCR products were separated on 6% sequencing gels (GIBCO BRL) with the LI-COR gel electrophoresis apparatus in 1× TBE buffer at a constant 40 W (1100 V and 40 mA) and 40°C and were detected using the LI-COR Global Edition IR2 system.

Linkage Analysis

Linkage among the SSR markers was analyzed using MapMaker/EXP 3.0 software. The Kosambi mapping function was used to calculate map distances. A LOD threshold of 4.0 and a maximum distance of 30 cM were used to group and order the SSR markers. The linkage between the resistance loci and the SSR markers was established by placing the resistance loci on the linkage map using a relative LOD threshold of 2.0.

Results and Discussion

Disease resistance tests with an isolate of race 1 identified 19 F2 lines with a resistant homozygous genotype, 20 with a susceptible homozygous genotype, and 41 with a heterozygous genotype. Tests with an isolate of race 35 identified 22 F2 lines with a resistant homozygous genotype, 13 with a susceptible homozygous genotype, and 46 with a heterozygous genotype. The chi-squared tests indicated that observed segregation ratios had a good fit to a 1:2:1 ratio with a type I error rate of 0.9632 (χ2 = 0.075) and 0.1743 (χ2 = 3.494), respectively (Table 1). The chi-squared test for independent segregation strongly suggested linkage between the two genes with a type I error rate of 0.0000 (χ2 = 58.6) (Table 1). Linkage analysis of resistance to isolates of race 1 and race 35 suggested the map distance between Rsp1a and Rps7 was about 11.5 cM with a LOD score of 14.33. This linkage estimate is similar to that reported by Anderson and Buzzell (1992) (Figure 1B) when their genetic distance between Rsp1a and Rps7 of 12.5 ± 2.7 map units is converted to 14.6 ± 3.0 cM. The difference between the two distances is within the standard deviation caused by random error. No nonallelic interactions were found for resistance (alive/total) to either race by the SAS GLM procedure (P = .2503 for resistance to race 1 and P = .6414 for resistance to race 35).

Figure 1.

Locations of Rps1 and Rps7 on linkage group MLG N (Cregan et al. 1999) with references to the previous studies. (A) Linkage map for Rps1a and Rps7 from this study. (B) Linkage between Rps1a and Rps7 according to the study of Anderson and Buzzell (1992). (C) Linkage between Rps1a and RFLP markers reported by Diers et al. (1992). (D) Linkage between Rps7 and RFLP and SSR markers reported by Lohnes and Schmitthenner (1997). (E) Linkage map consisting of RFLP and SSR markers reported by Cregan et al. (1999) using the mapping population of the University of Nebraska. (F) Linkage map consisting of RFLP and SSR markers reported by Cregan et al. (1999) using the mapping population of USDA/Iowa State University. (E) and (F) were simplified by taking away some markers from the linkage groups.

Table 1.

Monogenic and digenic segregation of resistance to Phytophthora sojae isolates of race 1 and race 35 in the F2 soybean population (OX281/Mukden) consisting of 81 lines

aChi-squared probability for observed fit to an expected digenic ratio of 1:2:1:2:4:2:1:2:1.

bNo result for race 1 on one F2 line.

Table 1.

Monogenic and digenic segregation of resistance to Phytophthora sojae isolates of race 1 and race 35 in the F2 soybean population (OX281/Mukden) consisting of 81 lines

aChi-squared probability for observed fit to an expected digenic ratio of 1:2:1:2:4:2:1:2:1.

bNo result for race 1 on one F2 line.

A total of 25 PCR primer pairs were tested for polymorphism in the mapping population by screening the primers against the two parents, OX281 and Mukden. These PCR primers were previously identified to amplify SSR marker loci on linkage group MLG N (Cregan et al. 1999). These SSR loci are known to be linked with Rps7 (Lohnes and Schmitthenner 1997) and hence with Rps1 (Anderson and Buzzell 1992). Eleven of the 25 SSR markers were polymorphic between the two parents. Chi-squared goodness-of-fit tests showed none of the 11 SSR loci deviated from the 1:2:1 (AA:AB:BB) ratio at a type I error rate of 0.05 (Table 2). Ten of the 11 polymorphic markers were mapped into one linkage group covering 114.3 cM (Figure 1A). One SSR marker, Satt022, was too loosely linked to be placed into the same linkage group. When mapped with the 10 SSR markers, the two resistance genes, Rps1a and Rps7, were placed at two different loci 13.8 cM apart with LOD scores of 2.88 and 9.16, respectively. Rps1a was flanked by Satt159 (0.7 cM) and Satt009 (3.2 cM). Rps7 was flanked by Satt009 (10.6 cM) and Satt125 (29.1 cM) (Figure 1A).

Table 2.

Segregation of the 11 SSR loci in the F2 population (OX281/Mukden) consisting of 81 lines and tests for distortion using chi-squared tests

Genotype AA is homozygous for the OX281 allele, genotype BB is homozygous for the Mukden allele, and genotype AB is heterozygous. N is the total number of plants that had valid scores; χ2 is the chi-squared statistic; df is degree of freedom for the chi-squared test; P is the type I error rate for the chi-squared test. The expected ratio of AA:AB:BB for each loci is 1:2:1.

Table 2.

Segregation of the 11 SSR loci in the F2 population (OX281/Mukden) consisting of 81 lines and tests for distortion using chi-squared tests

Genotype AA is homozygous for the OX281 allele, genotype BB is homozygous for the Mukden allele, and genotype AB is heterozygous. N is the total number of plants that had valid scores; χ2 is the chi-squared statistic; df is degree of freedom for the chi-squared test; P is the type I error rate for the chi-squared test. The expected ratio of AA:AB:BB for each loci is 1:2:1.

The genetic distance between and the order of the SSR markers were similar to those reported by Cregan et al. (1999) using the mapping population of USDA/Iowa State University except that our marker order for Satt152 and Satt159 was reversed (Figure 1F). However, our order was identical to that found in Cregan et al. (1999) using the mapping population of the University of Nebraska (Figure 1E). The genetic distances between the SSR markers were about the same, but overall the distances calculated based on our study were 13.5 cM longer for the interval spanning Satt152/Satt159 and Satt257 (Figure 1A,F).

The location of Rps1 was comparable to that obtained by Diers et al. (1992) (Figure 1C), but the location of Rps7 was not in agreement with that reported by Lohnes and Schmitthenner (1997) (Figure 1D), neither by their inference nor by integration of separate studies. Diers et al. (1992) reported that Rps1 was 6 cM from RFLP marker A280-1 (Figure 1C), while the distance was estimated to be 3.2 cM between Rps1 and Satt009 and 9.8 cM between Rps1 and A280-1 based on our results and the molecular linkage maps constructed by Lohnes and Schmitthenner (1997) and Cregan et al. (1999). The distance between Rps7 and Satt009 reported by Lohnes and Schmitthenner (1997) was 28.5 cM (Figure 1D), but the distance calculated with our data was 10.6 cM. Moreover, the orientation of the gene and molecular markers was opposite. Their results showed that Rps7 was at the end of linkage group MLG N (Figure 1D), but our results suggested that Rps7 was between Satt009 and Sat033. In our MapMaker analysis, Rps7 was placed with a relative LOD score of 9.16, which strongly suggested preference for this location than the next most likely location. No data were available on the likelihood of linkage for the study of Lohnes and Schmitthenner (1997).

The linkage information in our study could be useful for map-based cloning and marker-assisted selection for genes at the Rps1 and Rps7 loci. Rps1a was flanked by Satt159 (0.7 cM) and Satt009 (3.2 cM). Satt159 may be used to screen yeast artificial chromosome (YAC) or bacterial artificial chromosome (BAC) libraries to identify the YAC clone or BAC clone containing the Rps1a gene. Rps1a and Satt159 may possibly locate in the same YAC clone or BAC clone if the clone is large enough and Rps1a is located in a gene-rich region where recombination is more frequent. Satt159 and Satt152 may be used to genotype the progeny for selection of the Rps1a gene in soybean breeding programs. The accuracy of genotyping was theoretically estimated to be 99.3% and 99.985% for marker-assisted selection using only Satt159 and using both Satt159 and Satt152, respectively, to genotype the progeny on Rps1a. Rps7 was flanked by Satt009 (10.6 cM) and Satt125 (19.1 cM). Map-based cloning of Rps7 using either marker alone will be inefficient. However, the accuracy of genotyping was theoretically estimated to be 98.5% for dual-marker-assisted selection using both Satt009 and Satt125 to genotype progeny on Rps7. Satt530 may be an SSR marker tightly linked to Rps7 (Figure 1A,F), but it was not polymorphic in our mapping population. The linkage of Rps7 and Satt530 would be worthy of testing.

Corresponding Editor: Susan Gabay-Laughhan

The authors thank Margaret Haffner, Chuck Meharg, Elaine Lepp, and Bailing Zhang for technical assistance.

References

Anderson TR and Buzzell RI,

1992

. Inheritance and linkage of the Rps7 gene for resistance to Phytophthora rot of soybean.

Plant Dis

76

:

958

–959.

Athow KL and Laviolette FA,

1982

. Rps6, a major gene for resistance to Phytophthora megasperma f. sp. glycinea in soybean.

Phytopathology

72

:

1564

–1567.

Athow KL, Laviolette FA, and Mueller EH,

1980

. A new major gene for resistance to Phytophthora megasperma var. sojae in soybean.

Phytopathology

70

:

977

–980.

Bernard RL, Smith PE, Kaufmman MJ, and Schmitthenenner AF,

1957

. Inheritance of resistance to phytophthora root and stem rot in the soybean.

Agron J

49

:

391

.

Buzzell RI and Anderson TR,

1992

. Inheritance and race reaction of a new soybean _Rps_1 allele.

Plant Dis

76

:

600

–601.

Buzzell RI and Anderson TR,

1981

. Another major gene for resistance to Phytophthora megasperma var. sojae in soybean.

Soybean Genet Newslett

18

:

30

–33.

Cregan PB, Jarvik T, Bush AL, Shoemaker RC, Lark KG, Kathler AL, Kaya N, VanToai TT, Lohnes DG, Chung J, and Specht JE,

1999

. An integrated genetic linkage map of the soybean genome.

Crop Sci

39

:

1464

–1490.

Demirbas A, Rector BG, Lohnes DG, Fioritto RJ, Graef GL, Cregan PB, Shoemaker RC, and Specht JE,

2001

. Simple sequence repeat markers linked to the soybean Rps genes for phytophthora resistance.

Crop Sci

41

:

1220

–1227.

Diers BW, Mansur L, Imsande J, and Shoemaker RC,

1992

. Mapping Phytophthora resistance loci in soybean with restriction fragment length polymorphism markers.

Crop Sci

32

:

377

–383.

Kilen TC, Hartwig EE, and Keeling BL,

1974

. Inheritance of a second major gene for resistance to phytophthora rot in soybeans.

Crop Sci

14

:

260

–262.

Lohnes DG and Schmitthenner AF,

1997

. Position of the Phytophthora resistance gene _Rps_7 on the soybean molecular map.

Crop Sci

37

:

555

–556.

Mueller EH, Athow KL, and Laviolette FA,

1978

. Inheritance of resistance to four physiological races of Phy-tophthora megasperma var. sojae.

Phytopathology

68

:

1318

–1322.

Polzin KM, Lohnes DG, Nickell CD, and Shoemaker RC,

1994

. Integration of _Rps_2, Rmd, and _Rj_2 in linkage group J of the soybean molecular map.

J Hered

85

:

300

–303.

Schmitthenner AF, 1999. Phytophthora rot. In: Compendium of soybean diseases, 4th ed (Hartman GH, Sinclair JB, and Rupe JC, eds). St. Paul, MN: American Phytopathological Society; 39–42.

Shoemaker RC and Specht JE,

1995

. Integration of the soybean molecular and classic genetic linkage groups.

Crop Sci

35

:

436

–446.

Yu K, Park SJ, and Poysa V,

1999

. Abundance and variation of microsatellite DNA sequences in beans (Phaseolus and Vigna).

Genome

42

:

27

–34.