cell biology & molecular genetics - PubAg - USDA

CELL BIOLOGY & MOLECULAR GENETICS Simple Sequence Repeat Markers Linked to the Soybean Rps Genes for Phytophthora Resistance A. Demirbas, B. G. Rector, D. G. Lohnes, R. J. Fioritto, G. L. Graef, P. B. Cregan, R. C. Shoemaker, and J. E. Specht* ABSTRACT

ease is most effectively managed by planting resistant cultivars (Athow, 1987). Naturally occurring resistance to Phytophthora root rot exists in soybean. Monogenic race-specific resistance to the causal organism is known to be controlled by 13 dominant alleles at seven soybean loci, designated Rps1 (Bernard et al., 1957), Rps2 (Kilen et al., 1974), Rps3 (Mueller et al., 1978), Rps4 (Athow et al., 1980), Rps5 (Buzzell and Anderson, 1981), Rps6 (Athow and Laviolette, 1982), and Rps7 (Anderson and Buzzell, 1992). The Rps1 and Rps7 loci are linked (Lohnes and Schmitthenner, 1997), as are the Rps4 and Rps5 loci (Diers et al., 1992). By means of DNA marker technology (Botstein et al., 1980; Williams et al., 1990; Akkaya et al., 1992; Vos et al., 1995), marker-dense genetic linkage maps have been constructed in many crop species (O’Brien, 1993), including soybean (Shoemaker and Specht, 1995; Keim et al., 1997; Cregan et al., 1999). Plant breeders can use molecular markers to select indirectly individuals in segregating populations that carry a gene for a favorable trait if a tight linkage exists between a marker locus and the genetic locus controlling that trait (Tanksley et al., 1989). Marker-assisted selection (MAS) allows the breeder to bypass laborious and/or costly phenotypic screens. Moreover, if two closely linked marker loci are present on either side of the desired locus, the retention of donor DNA in NILs of cultivars with the introgressed gene could be minimized (Young and Tanksley, 1989). Bulked segregant analysis is an effective strategy for identifying linkages between genetic markers and qualitative traits (Michelmore et al., 1991). A population of F2 individuals segregating for two alleles of a single gene (or marker) can be sorted into two classes comprised of the contrasting homozygous genotypes. If the DNA bulks corresponding to those two classes display a polymorphism for a genetic marker, linkage between the marker and the segregating locus can be inferred. The number of F2 individuals that should be composited in each bulk is dependent upon the number of false inferences of linkage that the investigator is willing to tolerate. In any event, putative linkages must eventually be verified by a genetic marker analysis of F2 individuals. However, verification of a putative linkage does not necessarily require a complete F2 data set. Allard (1956)

Simple sequence repeat (SSR) markers with linkages to the Rps1, Rps2, Rps3, Rps4, Rps5, and Rps6 loci that govern soybean [Glycine max (L.) Merr.] resistance to Phytophthora root rot (caused by Phytophthora megasperma Drechs. f. sp. glycinea Kuan and Ervin) are desired. Near-isogenic lines (NILs) of Clark or Williams, homozygous resistant (RpsRps ) at just one of those Rps loci, were mated to a NIL of Harosoy homozygous susceptible (rpsrps ) at all six loci. From the 100 to 120 F2:3 progenies per mating, 20 F3 seedlings were evaluated for resistance (R) or susceptibility (S) following inoculation with the race of P. megasperma affected by the segregating Rps allele. About 15 RpsRps and 15 rpsrps F2 individuals were used to construct contrasting DNA bulks. Presumptive linkage (i.e., SSR marker polymorphism between two bulks) was confirmed or refuted by SSR assay of 15 to 40 F2 individuals within each homozygous class. Recombination values were maximum likelihood estimates from the SSR allelic segregation data of both classes, although the rpsrps class was less prone to phenotypic classification error. SSRs on linkage groups (LGs) N, J, F, and G were identified with linkages to Rps1, Rps2, Rps3, and Rps4, respectively. A skewed R:S segregation in the Rps5 population precluded detection of linked SSRs. The Rps6 locus, whose map position was heretofore unknown, was linked with three SSRs in a region of LG-G that contains Rps4 and Rps5. SSR–Rps linkages of P ⬍ 0.05 could only be identified for the Rps1 alleles because of a paucity of SSR markers and/or parental monomorphism in the genomic regions surrounding other Rps loci.

P

hytophthora root rot can cause severe economic losses in soybean production (Diers et al., 1992). This root rot disease is favored by wet conditions, and tends to be prevalent in soils whose poor drainage leads to retention of water on the soil surface. Symptoms include yellowing and wilting of the leaves and brown discoloration of the lower stem and branches. The disA. Demirbas, Black Sea Agric. Res. Inst., P.K. 39, Samsun, Turkey; B.G. Rector, USDA, ARS, SAA, CGBRU, P.O. Box 748, Tifton, GA 31793; D.G. Lohnes and R.J. Fioritto, Dep. of Hort. & Crop Sci., Ohio Agric. Res. & Development Center, Ohio State Univ., 1680 Madison, Ave., Wooster, OH; P.B. Cregan, USDA, ARS, Bldg. 006, Room 100, BARC-West, 10300 Baltimore Ave., Beltsville, MD; R.C. Shoemaker, USDA, ARS, Corn Insect and Crop Genetics Research Unit, Dep. of Agronomy, Iowa State Univ., Ames, IA 50011; G.L. Graef and J.E. Specht, Dep. of Agronomy, Univ. of Nebraska, Lincoln, NE 68583-0915. Published as Paper no. 13137, Journal Series, Nebraska Agric. Res. Div. Project no. 12-194. Research supported by state and federal funds appropriated to the Agric. Res. Div. and Univ. of Nebraska, and by grants received from the United Soybean Board, the Nebraska Soybean Development, Utilization, and Marketing Board, and the Ohio Soybean Council. Received 18 Sept. 2000. *Corresponding author ( [email protected]).

Abbreviations: MAS, marker-assisted selection; SSR, simple sequence repeat; NIL, near-isogenic line; BSA, bulked-segregant analysis; LG, linkage group; RFLP, restriction fragment length polymorphism.

Published in Crop Sci. 41:1220–1227 (2001).

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DEMIRBAS ET AL.: SSR MARKERS LINKED TO THE SOYBEAN Rps GENES

published maximum likelihood equations for estimating linkage (i.e., a recombination value and its standard error) from incomplete F2 data. One common example of an incomplete F2 data set is the segregation of a codominant marker within a homozygous genotypic class produced by another marker. For MAS applications, the most desirable markers would map to a single genomic position but each marker would have many alleles, which would increase the probability of marker polymorphism. SSR markers are single-locus markers for which the variation in the number of tandem two or three base pair repeats offers great potential for multiple allelism (Cregan et al., 1999). SSR technology is also based on the polymerase chain reaction (PCR), which makes it accessible to most plant breeders. Moreover, the DNA amplicons of SSR markers are often resolvable on high-resolution agarose gels stained with ethidium bromide; offering the breeder using MAS a convenient alternative to polyacrylamide gels and radio isotopes. The objective of this research was to use bulkedsegregant analysis to identify soybean SSR markers tightly linked to each of the six known Phytophthora resistance genes: Rps1 to Rps6. SSR linkages have already been identified for the other known resistance gene, Rps7, on the basis of its in situ segregation in one of the mapping populations used in the construction of the soybean SSR map (Lohnes and Schmitthenner, 1997; Cregan et al., 1999). MATERIALS AND METHODS Plant Materials The 11 parents used in the matings are described in Table 1. Seed of these parental NILs was obtained from the curator of the U.S. soybean germplasm collection (R.L. Nelson, USDAARS, Urbana, IL). Ten of these 11 parental NILs originated as BC5-derived S1 progeny selections from the matings of a recurrent parental cultivar (Clark or Williams) with donor parent sources of 10Rps alleles representing six different loci (i.e., Rps1, Rps1b, Rps1c, Rps1d, Rps1k, Rps2, Rps3, Rps4, Rps5, and Rps6). The cultivar Harosoy is homozygous rpsrps (i.e., susceptible) at all six loci. A Harosoy NIL possessing the introgressed maturity gene E2 (located on soybean linkage group O) was chosen for the matings, because its flowering and maturity dates were closer to those of the Clark and Williams NILs. Genetic markers were used to confirm the expected heterozygosity in the F1 plants produced from matings of the male parent, Harosoy-E2, to each of the 10 Phytophthora-resistant parental NILs. The F1 plants were selfed to produce populations that totaled to about 100 to 120 F2 plants. In each F2 population, only the specific Phytophthora resistance allele and its susceptible counterpart were segregating at the given locus. F2 plants in each population were grown in a nursery in which Phytophthora root rot had never been observed, and none was observed in this F2 generation. The F2 plants were selfed and threshed individually to produce F2:3 seed progenies.

Phytophthora Resistance Testing F2:3 phenotypic data was obtained by inoculating F3 individuals in each F2:3 family with the race of P. megasperma corresponding to the resistance allele segregating in the population.

Table 1. Phytophthora-resistant parents mated to a common susceptible parent to enable segregation of the indicated allele for resistance, observed numbers of the three F2 genotypes based on F2:3 phenotyping, and Chi-square tests of the goodness-of-fit of those observed numbers to a 1:2:1 ratio. F2 Genotype Resistant parent†

Allele RpsRps Rpsrps rpsrps Total no.

Clark L61-422(L7) Clark L77-2061 Clark L75-3901 Williams L93-3312 Clark L77-2015 Clark L76-2060 Williams L83-705 Williams L85-2352 Williams L85-3059 Williams L89–1581

Rps1 Rps1b Rps1c Rps1d Rps1k Rps2 Rps3 Rps4 Rps5 Rps6

29 35 40 04 44 15 35 26 14 26

no 37 48 34 24 52 71 37 51 41 58

18 30 21 19 15 29 17 23 67 35

84 113 95 47 111 115 89 100 122 89

␹2 4.07 3.00 15.27** 9.60** 15.59** 9.75** 9.81** 0.22 59.16** 1.44

**, Denotes statistical significance of the Chi-square value (␣ ⫽ 0.01). † Each Rps parent was mated to Harosoy–E2 (L62-4584), which was homozygous rps at all six loci.

A hypocotyl inoculation technique, using mycelia, was used to classify the plants for reaction to P. megasperma (Schmitthenner et al., 1994). The phenotypic assay was conducted in the following manner. Seeds of each F2:3 progeny were planted 10 to a pot. One pot of each parental genotype (also 10 seeds/ pot) was included as a control for each group of 20 progeny pots. Seedlings were inoculated 10 d after planting. The number of living, diseased, and dead seedlings was recorded 3 to 5 d after inoculation. If all 10 F3 seedlings in a pot were deemed susceptible, the F2 progenitor of those progeny was classified as rpsrps. The probability (␣) of observing only rpsrps F3 progeny from a Rpsrps F2 progenitor is qn ⫽ 10⫺6, based on n ⫽ 10 F3 seedlings and a q ⫽ 0.25 probability of a rpsrps from a heterozygous F2 (Hanson, 1959). If the 10 F3 seedlings segregated for resistance and susceptibility, the F2 progenitor was classified as Rpsrps. However, if all 10 F3 seedlings were deemed resistant, then another 10 F3 seedlings were planted and inoculated, and if all 20 F3 seedlings proved to be resistant, then the F2 progenitor was classified as RpsRps. The probability (␣) of observing only Rps—F3 progeny from a Rpsrps F2 progenitor, is qn ⫽ 0.0032, based on n ⫽ 20 F3 seedlings and a q ⫽ 0.75 probability of RpsRps or Rpsrps from a heterozygous F2. Inoculation of the resistant parents and checks provided periodic assessment of RpsRps seedling death arising from mechanical inoculation injury. The frequency of these was near zero in all screenings. Inoculation of the susceptible parents and checks provided periodic assessment of rpsrps seedling survival (i.e., disease escape) arising from variation in inoculation efficacy or inoculum pathogenicity. Any screening run in which the frequency of disease escapes was in excess of 5% was repeated using remnant F3 seed.

DNA Isolation Leaf tissue was collected in the field from each parent and from approximately 20 to 25 random F3 plants from the F2:3 progeny rows that had been classified as homozygous dominant RpsRps or recessive rpsrps in the greenhouse screening bioassay described above. No tissue was collected from the F2:3 rows classified as heterozygous. The leaf tissue was transported to the laboratory on dry ice, frozen, and stored at ⫺20⬚C. The frozen leaf tissue was eventually lyophilized and ground to a fine powder that was also stored at ⫺20⬚C until DNA extraction could be performed. DNA was extracted using a protocol modified from that of Saghai-Maroof et al.

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(1984). After DNA extraction and its resuspension in TE, the DNA samples were diluted to 20 ng/mL for SSR analysis.

Bulked Segregant Analysis (BSA) and SSR Marker Electrophoresis/Staining Paired primers specific for the amplification of each SSR were used. This study was initiated prior to the integration of the soybean SSR and restriction fragment length polymorphism (RFLP) maps (Cregan et al., 1999), when the final map positions of the SSR markers were not yet known, so parental screening for SSR polymorphism was commenced using all available SSRs. After SSR/RFLP map integration, parental SSR assays were limited mainly to those SSRs that were in the same linkage groups as the RFLP markers that Diers et al. (1992) had earlier reported were linked to Rps1 thru Rps5. Because of the multi-locus nature of the RFLP markers linked to Rps4 and Rps5, SSRs linked to each locus of those RFLP markers were used to assay the Rps4 and Rps5 populations. Parental SSR assays of the Rps6 locus, whose map position was unknown, continued until an SSR linkage was found, but thereafter parental assays were limited to SSRs in the same linkage group. Polymorphism at any given SSR locus was detected according to the procedures of Akkaya et al. (1992) and Rongwen et al. (1995), except that the DNA amplicons were resolved on high resolution agarose gels instead of polyacrylamide gels, and were visualized with ethidium bromide staining instead of 32P labeling. A 10-␮L PCR reaction contained 50 ng of genomic DNA (parental, F2 bulk, or F2 individual), 0.1 ␮M of each primer, 5⫻ Reaction Buffer (250 mM Tris), 0.6 units of AmpliTaq DNA Polymerase (Perkin-Elmer Corporation, Norwlak, CT), 2.5 ␮M dNTPs. Each sample was covered with 10 to15 ␮L of light mineral oil and subjected to 31 cycles of denaturing (25 s at 94⬚C); annealing (25 s at 47⬚C); and extension (25 s at 68⬚C) in a DNA thermo-cycler, followed by a final extension step (3 min at 72⬚C) and incubation at 4⬚C. After PCR amplification, products were separated by electrophoresis by means of 5% (w/v) agarose gels stained with ethidium bromide and photographed under ultraviolet light. The contrasting DNA bulks were constructed by combining equal amounts of DNA collected from about 15 F2:3 progenies of the 15 to 40 available within each population as either homozygous RpsRps, or homozygous rpsrps. Although 15 individuals per bulk is larger than the recommendation of eight by Michelmore et al. (1991), a lower experimentwise rate of false inferences of putative linkage was preferred for the present study. The SSR marker assays were conducted in three stages. First, the parents of each cross were screened for polymorphism with SSR markers. Second, the informative (i.e., parentally polymorphic) SSRs were used to screen the contrasting F2 bulks to determine if the parental polymorphism was fully or partially maintained in those bulks. If so, that finding was considered presumptive evidence of linkage. Third, individuals within the F2 homozygous resistant (RpsRps ) class, and within the F2 homozygous susceptible (rpsrps ) class, were evaluated with any SSR that displayed a polymorphism between the bulks.

of the SSR marker alleles (i.e., the Harosoy–E2 (rps ) parent was designated as the source of the SSR ‘B’ allele). Chi-square analyses were also performed to determine if the genotypic segregation of an SSR marker within a given F2 homozygous class (either RpsRps or rpsrps ) differed significantly from the 1AA:2AB:1BB ratio which would be expected for an SSR locus not linked to the Rps/rps locus (Table 2). In addition, the ratio of AA to BB marker genotypes, when their numbers were summed over the contrasting homozygous classes (i.e., RpsRps and rpsrps ), was evaluated to determine if it fit the 1:1 ratio expected if the SSR andRps loci were independent. Because of limited seed amounts, the number of homozygous F2:3 progenies for which DNA was available for SSR genotyping (Table 2) was sometimes slightly less than the number of homozygous F2:3 progenies that were identified in the phenotypic bioassay (Table 1). The maximum likelihood method was used to derive a recombination value (p ) for each putative SSR-Rps linkage. Maximum likelihood equations for estimating linkage from different kinds of incomplete F2 data were published by Allard (1956). His Eq. [16], which is designed for segregation of the three genotypes (AA, AB, BB) of a codominant marker within the homozygous recessive class of another marker, is as follows:

l ⫻ [2/(p ⫺ 1)] ⫹ m ⫻ [(1 ⫺ (2 ⫻ p))/(p ⫻ (1 ⫺ p))] ⫹ n ⫻ [2/p] ⫽ 0 where l, m, and n are the respective observed numbers of the AA, AB, and BB genotypes detected among the F2 homozygous recessive (i.e., rpsrps ) individuals, and p is the recombination value. The DNA banding pattern of Harosoy–E2 (rps ) parent was always inferred to be the SSR genotype BB. After substitution of the observed values of l, m, and n in the above equation, a spreadsheet was used to search for the p value that would render the equation equal to zero. The resulting p value represents the most likely recombination value for the observed data. The above equation can also be used for the segregation of AA, AB, and BB genotypes within the homozygous dominant (i.e., RpsRps ) resistant class, but upon rendering the equation equal to zero, the solution is (1 ⫺ p ). The standard error (SE) for a p estimate obtained with equation #16 (Allard, 1956) was calculated as follows:

SEp ⫽ [1/(x ⫻ ip)]1/2 where x is the total number of F2 individuals examined, and

ip ⫽ 1/[2p ⫻ (1 ⫺ p)] which is a mathematical formulation reflecting the information value associated with the SSR genotyping of an F2 homozygous recessive (or dominant) at the given Rps locus. In rare cases, an SSR behaved as a dominant marker (i.e., absence of the Harosoy amplicon), perhaps because one of the primers did not anneal completely with the Harosoy parent locus. To estimate linkage when only the dominant (amplicon present) and recessive (amplicon absent) genotypes of the SSR marker were observable within the homozygous recessive class of a repulsion phase parental mating, Eq. [7] (Allard, 1956) was used:

Linkage Analysis

c ⫻ (⫺2p/1 ⫺ p2) ⫹ d ⫻ (2/p) ⫽ 0

Chi-square analyses were conducted to determine if the observed segregation at each givenRps locus fit the expected F2 segregation ratio of 1RpsRps:2Rpsrps:1rpsrps (Table 1) or, if that ratio was not evident, whether the observed segregation fit an expected 3Rps—:1rpsrps ratio. EachRps ⫻ rps mating was treated as an AA ⫻ BB mating with respect to the coding

where c and d correspond respectively to the numbers of SSR dominant (A_) and recessive (BB) individuals within the homozygous recessive susceptible (rpsrps ) class. This equation is not symmetrical, so for the coupling phase mating, (1 ⫺ p ) must be substituted for p in the above equation and the signs reversed (Allard, 1956). The standard error of the linkage

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Table 2. For each mating involving the indicated Rps allele, the number of SSR loci tested, the percentage that were parentally polymorphic, and the number of resistant RpsRps and susceptible rpsrps homozygotes in the F2 population eventually assayed with those polymorphic SSRs. Rps allele Rps1 Rps1b Rps1c Rps2 Rps3 Rps4 Rps6

SSRs tested

Polymorphic SSRs

no 228 228 228 228 242 251 420

% 30.7 30.7 28.5 27.6 25.2 16.7 19.3

Resistant RpsRps

Susceptible rpsrps

Table 3. Linkage estimates obtained for the SSR markers segregating in the Rps1 ⫻ rps1 population. Map distance cM† –

no 27 29 37 14 34 25 19

18 19 20 26 17 23 32

0.5

1.0

2.2

estimate is calculated in the same manner as described previously, except that an information value appropriate for Eq. [7] must be used:

ip ⫽ 1/(1 ⫺ p2). In this study, a putative SSR–Rps linkage was considered likely if the calculated p value, plus its standard error, totaled to less than 0.40, and quite likely if more than one SSR in a localized region of a particular linkage group was linked to a given Rps locus. For each SSR–Rps linkage, we report a p value and standard error computed from each homozygous class, as well as a mean p value and standard error computed from the SSR segregation data pooled over both homozygous classes.

RESULTS Phenotypic data relative to the screening of F2:3 progeny for Phytophthora resistance are presented in Table 1. Chi-square analyses indicated that the Rps1, Rps1b, Rps4, and Rps6 populations had satisfactory fits to a monogenic ratio of 1RpsRps:2Rpsrps:1rpsrps. The remaining six populations deviated significantly from the expected 1:2:1 ratio, but satisfactory fits to a 3Rps: 1rpsrps ratio were observed for the Rps1c (␹2 ⫽ 0.07; P ⫽ 0.79), Rps2 (␹2 ⫽ 0.003; P ⫽ 0.96), and Rps3 (␹2 ⫽ 1.35; P ⫽ 0.24) populations. Although the classification of the resistant F2:3 progenies in these three populations into homozygous (RpsRps) and heterozygous (Rpsrps) classes was apparently not error-free, the Chi-square tests suggested that the observed fraction of susceptible F2:3 progenies was close to that expected for homozygous recessive (rpsrps) progenies, and thus it would be suitable for use in linkage analysis. Relative to the remaining populations, a significant deviation from a 3:1 ratio was observed for Rps1d (␹2 ⫽ 5.17, P ⫽ 0.02), Rps1k (␹2 ⫽ 7.21, P ⫽ 0.007), and Rps5 (␹2 ⫽ 56.66, P ⬍ 0.0001). The segregational skewing in these three populations did not appear to be due to a reversal of dominant and recessive alleles, since significant non-fits to a 1RpsRps:3 (2Rpsrps: 1rpsrps) ratio were also evident for Rps1d, Rps1k and Rps5 (␹2 ⫽ 9.21, 12.68, and 11.90, respectively). Because monogenic segregation was not verifiable, these three populations were not suitable for linkage analyses. This was unfortunate with respect to identifying SSRs linked to the Rps5 locus. However, Rps1d and Rps1k are by definition allelic with Rps1, Rps1b, and Rps1c, so any SSR linked to the latter three should also be linked to the first two. The percentage of SSR markers that were polymorphic

1.6

44.0

25.7

SSR locus

Plant Rps genotype

Plant SSR genotype AA

AB

BB

16 0

9 1

2 17

19 0

5 1

3 17

25 1

2 0

0 17

13 0

12 0

2 18

12 0

13 0

3 18

10 6

12 7

5 5

9 7

11 6

7 5

Recombination P ⫾ SE

no Satt159 Rps1Rps1 rps1rps1 Mean Satt152 Rps1Rps1 rps1rps1 Mean Satt009 Rps1Rps1 rps1rps1 Mean Satt530 Rps1Rps1 rps1rps1 Mean Satt584 Rps1Rps1 rps1rps1 Mean Satt237 Rps1Rps1 rps1rps1 Mean Satt022 Rps1Rps1 rps1rps1 Mean

0.24 0.03 0.16 0.20 0.03 0.13 0.04 0.06 0.04 0.30 0.00 0.18 0.34 0.00 0.21 0.41 0.53 0.46 0.46 0.56 0.50

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.12 0.06 0.08 0.11 0.05 0.07 0.05 0.08 0.04 0.12 0.00 0.08 0.13 0.00 0.08 0.13 0.17 0.11 0.14 0.17 0.11

† Linkage Group N map distance between the indicated SSR and the SSR immediately above (from Cregan et al., 1999). Other Linkage Group N SSRs were not parentally polymorphic in this population.

between the Harosoy–E2 parent and the Clark (Rps1 to Rps2 loci) or Williams (Rps3, Rps4, and Rps6 loci) parents ranged from 16.7 to 30.7%, averaging about 25.5% (Table 2). SSR polymorphism was generally lower in the Williams ⫻ Harosoy matings probably because the cultivar A.K. (Harrow), a parent of Harosoy, is nearly identical to the cultivar Illini, a great-grandparent of Williams.

Resistance Locus Rps1 Of the 228 SSRs tested on the Harosoy–E2 and Clark– Rps1 parents, 70 (30.7%) were polymorphic (Table 2). These SSRs were distributed mostly at random over the soybean genetic map. Of the 70 parentally polymorphic SSRs, seven (10%) map to Linkage Group (LG) N. In a prior linkage study involving RFLP markers, Diers et al. (1992) had positioned Rps1 in an RFLP linkage group then known as LG-K. In the recently published soybean SSR map (Cregan et al., 1999), that RFLP linkage group is now part of LG-N. Putative linkages between the Rps1 locus and the LG-N markers Satt159 and Satt152 were observed in the bulked segregant analysis. To confirm these putative linkages, SSR genotyping was performed on each of 27 homozygous resistant (Rps1Rps1) and 18 homozygous susceptible (rps1rps1) F2 individuals (Table 2). The mean recombination (p) between Satt159 and Satt152 and Rps1 was 0.16 and 0.13, respectively (Table 3). However, the linkage estimates based on the homozygous susceptible rps1rps1 individuals were much lower (for both SSRs, only one progeny was recombinant) when compared to those based on the homozygous resistant Rps1Rps1 individuals. The Rps1 population had satisfactory fits to the expected 1RpsRps:2Rpsrps:1rpsrps ratio (Table 1), and to the expected 3Rps-:1rpsrps ratio; however, the

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homozygous resistant and heterozygous classes did not fit the expected 1:2 ratio, suggesting that some heterozygous progenies were probably mis-scored as homozygous resistant progenies. If so, then the linkage computed from the homozygous susceptible class in Table 3 may be more reliable. Identical p ⫽ 0.03 estimates were derived from that class for the linkage of Rps1 to both Satt152 and Satt159, which is consistent with the tight linkage of the two SSR markers with each other (i.e., 0.5 cM in the map presented by Cregan et al., 1999). SSR marker Satt009 displayed a polymorphism in the Rps1/rps1 bulks. Satt009 is located about 1.5 cM below Satt152 on LG-N. In this paper, the terms below and above refer to the vertical order of markers in the linkage groups published by Cregan et al. (1999). However, the Satt009 primers, when used on a parental DNA composite designed to simulate genomic DNA of an F1 or F2 heterozygote, did not produce a heterozygous DNA banding pattern, or did so unpredictably and only infrequently. This anomaly could make Satt009 unsuitable for MAS. Tests of F2 individuals for linkage to SSR markers Satt530 and Satt584, which are located 3.2 and 4.8 cM, respectively, below Satt152 (Cregan et al., 1999) revealed that these two markers were tightly linked to Rps1 (Table 3). In fact, within the homozygous susceptible rps1rps1 class, no recombinants were observed for either SSR, suggesting tight linkage. Within the homozygous resistant Rps1Rps1 class, many recombinants were observed but, as was noted before, this class may have been contaminated with heterozygous Rps1rps1 individuals. Segregation data were also obtained for the polymorphic SSRs Satt257 and Satt022, located 61.5 and 70.5 cM, respectively, below Satt584 (Cregan et al., 1999). As expected, these two SSR markers segregated independently of the Rps1 locus (Table 3). Segregation of the Rps1b allele fit the expected 1:2:1 ratio, but segregation of the Rps1c allele did not (Table 1). Rps1c segregation did fit a 3:1 ratio (␹2 ⫽ 0.0737, P ⫽ 0.7860), so the homozygous recessive class was considered suitable for linkage analysis. The same 228 SSRs tested on the Rps1 population were used to screen the Rps1b and Rps1c populations. Seventy SSRs (30.7%) in the Rps1b population and 65 SSRs (28.5%) in the Rps1c population were polymorphic between the respective parents (Table 2). Several LG-N SSRs segregating in the Rps1 population were not segregating in the Rps1b and Rps1c populations, although Satt152, Satt530, and Satt584 did segregate in all three populations. Satt152 was linked to Rps1, Rps1b, and Rps1c with respective mean p values of 0.13 (Table 3), 0.16 (Table 4), and 0.24 (Table 5). Considering only the SSR genotypes in the homozygous recessive classes, the respective p values were 0.03 (Table 3), 0.09 (Table 4), and 0.15 (Table 5). Satt530 and Satt584 were also linked to Rps1b and Rps1c, but not as tightly as those two SSRs were linked with Rps1. For the homozygous recessive classes, the respective Satt530 and Satt584 linkages to Rps1, Rps1b, and Rps1c were 0.00 and 0.00 (Table 3), 0.12 and 0.20 (Table 4), and 0.14 and 0.21 (Table 5).

Table 4. Linkage estimates obtained for the SSR markers segregating in the Rps1B ⫻ rps1 population. Map distance cM† –

3.2

1.6

SSR locus

Plant Rps genotype

Satt152 Rps1bRps1b rps1rps1 Mean Satt530 Rps1bRps1b rps1rps1 Mean Satt584 Rps1bRps1b rps1rps1 Mean

Plant SSR genotype Recombination AA

AB

BB

18 0

no 8 5

3 24

20 2

5 2

4 21

17 1

9 9

3 18

P ⫾ SE 0.24 0.09 0.16 0.22 0.12 0.18 0.26 0.20 0.23

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.11 0.07 0.07 0.11 0.09 0.07 0.12 0.11 0.8


Resistance Locus Rps2 Sixty-three of 228 SSR markers (27.6%) were polymorphic between Harosoy–E2 and Clark–Rps2 (Table 2). Of these, only Satt547 on LG-J generated a polymorphism in the bulked segregant analysis. The mean linkage between Satt547 and Rps2 was 0.24 (Table 6). Satt287 was the only other parentally polymorphic LG-J SSR in the Rps2 population, but it is a substantial distance above of Satt547 (Table 2). SSR markers are scarce in the lower part of LG-J (Cregan et al., 1999). The moderate linkage of Rps2 with Satt547 was of insufficient intensity for use in MAS, but was useful in confirming the positioning of Rps2 onto LG-J of the integrated soybean SSR map (Cregan et al., 1999) based on linkage between SSR markers on LG-J and RFLP markers linked to Rps2 (Diers et al., 1992; Polzin et al., 1994).

Resistance Locus Rps3 Of 242 SSR markers screened, 61 (25.2%) detected polymorphism between the Harosoy–E2 and Williams– Rps3 parents (Table 2). Of these 61 SSRs, four showed differences in parental DNA band intensities between the two DNA bulks, suggesting linkage with Rps3. These four SSR markers were Satt374, SOYHSP176, Satt114, and Satt144. All map to LG-F (Cregan et al., 1999), which now includes the RFLP markers that Diers et al. (1992) reported to be linked to Rps3. Although a large Table 5. Linkage estimates obtained for the SSR markers segregating in the Rps1c ⫻ rps1 population. Map distance cM† –

3.2

1.6

SSR locus

Plant Rps genotype

Satt152 Rps1cRps1c rps1rps1 Mean Satt530 Rps1cRps1c rps1rps1 Mean Satt584 Rps1cRps1c rps1rps1 Mean

Plant SSR genotype Recombination AA

AB

BB

21 2

no 11 2

5 16

23 1

7 4

9 16

22 2

13 5

3 15

P ⫾ SE 0.28 0.15 0.24 0.32 0.14 0.26 0.25 0.21 0.23

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.11 0.11 0.08 0.11 0.11 0.08 0.10 0.12 0.08


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Table 6. Linkage estimates obtained for the SSR markers segregating in the Rps2 ⫻ rps2 population. Map distance

SSR locus

cM† –

Satt287

40.6

Satt547

Plant Rps genotype Rps2Rps2 rps2rps2 Mean Rps2Rps2 rps2rps2 Mean


AB

4 10

no 4 7

BB 5 9

10 4

3 7

1 17

Recombination P ⫾ SE 0.54 0.52 0.53 0.18 0.27 0.24

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.20 0.14 0.11 0.14 0.12 0.09

† Linkage Group J map distance between the indicated SSR and the SSR immediately above (from Cregan et al., 1999). Other Linkage Group J SSRs were not parentally polymorphic in this population.

number of SSR markers map to the same region (Cregan et al., 1999), parental polymorphism in this population was limited to just these four SSRs. A linkage analysis showed that SOYHSP176 and Satt114 were moderately linked to Rps3, with mean recombination values of 0.25 and 0.28, respectively (Table 7). The linkage estimates obtained from the homozygous recessive (rps3rps3) individuals were 0.18 and 0.12, respectively, and may be more accurate, given the possible error involved in distinguishing between the homozygous resistant and heterozygous progenies in this population (Table 1). Still, markers with tighter linkage will be required for effective MAS. Neither Satt144 nor Satt374 displayed any significant linkage to Rps3.

Resistance Locus Rps4 The Harosoy–E2 and Williams–Rps4 parents were screened with 251 SSRs, and 42 (17%) were polymorphic (Table 2). Of these 42, only Satt472 on LG-G displayed intensity differences between the parental DNA bands in the bulked DNA samples. Linkage analysis showed that Satt472 was linked to Rps4 with recombination value of 0.09 (Table 8). Diers et al. (1992) reported that RFLP markers T005 and A586 were linked to Rps4, but since these were multi-locus RFLPs, the LG assignment of Rps4 was ambiguous (Cregan et al., 1999). The direct linkage of Satt472 with Rps4 firmly establishes the positioning of Rps4 on LG-G. The intensity of the Table 7. Linkage estimates obtained for the SSR markers segregating in the Rps3 ⫻ rps3 population. Map distance cM† –

SSR locus

Plant Rps genotype

Satt374

Rps3Rps3 rps3rps3 Mean Rps3Rps3 rps3rps3 Mean Rps3Rps3 rps3rps3 Mean Rps3Rps3 rps3rps3 Mean

23.5

HSP176

2.2

Satt114

50.4

Satt144


AB

BB

13 2

no 13 7

8 8

19 2

11 2

4 13

15 0

13 4

6 13

11 2

16 7

7 8

Recombination P ⫾ SE 0.43 0.32 0.39 0.28 0.18 0.25 0.37 0.12 0.28 0.44 0.32 0.40

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.12 0.16 0.10 0.11 0.13 0.09 0.12 0.11 0.09 0.12 0.16 0.09

† Linkage Group F map distance between the indicated SSR and the SSR immediately above (from Cregan et al., 1999). Other Linkage Group F SSRs were not parentally polymorphic in this population.

Table 8. Linkage estimates obtained for the SSR markers segregating in the Rps4 ⫻ rps4 population. Map distance

SSR locus

cM† –

Satt109

35.5

Satt472

Plant Rps genotype Rps4Rps4 rps4rps4 Mean Rps4Rps4 rps4rps4 Mean


AB

BB

5 6

no 14 8

6 9

22 0

2 4

1 19

Recombination P ⫾ SE 0.52 0.44 0.51 0.08 0.09 0.08

⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.14 0.15 0.09 0.08 0.08 0.06

† Linkage Group G map distance between the indicated SSR and the SSR immediately above (from Cregan et al., 1999). Other Linkage Group G SSRs were not parentally polymorphic in this population.

linkage is borderline with respect to MAS, but it could be useful in applications where a low level of linkage phase reversal can be tolerated. The only other parentally polymorphic SSR on LG-G was Satt199, but it was too far above Rps4 to show linkage.

Resistance Locus Rps5 Diers et al. (1992) reported that RFLP marker T005 was linked to Rps5 as well as Rps4. The T005 marker maps to two loci, one on LG-B2 and another on LG-G. Given our SSR confirmation of an LG-G map position for Rps4 (see above), Rps5 would be expected to map to LG-G. Unfortunately, a skewed genotypic segregation in our Rps5 ⫻ rps5 population (Table 1) precluded an SSR-based confirmation of this genomic location for Rps5.

Resistance Locus Rps6 The map position of Rps6 was unknown prior to this investigation. Genotypic segregation in the Rps6 ⫻ rps6 population did not deviate significantly from the 1Rps6 Rps6:2Rps6rps6:1rps6rps6 ratio expected for a monogenic trait (Table 1). A total of 420 SSRs were tested on Harosoy–E2 and Williams–Rps6, and 81 (19.2%) were polymorphic (Table 2). Of these 81, just two, Satt472 and Satt191 on LG-G, displayed a noticeable difference in the intensities of the parental amplicons in the DNA bulks. SSR segregation data for 19 homozygous resistant and 32 homozygous susceptible classes generated linkage estimates of 0.23 and 0.22 between Rps6 and either Satt472 or Satt191, respectively (Table 9). Satt472 was Table 9. Linkage estimates obtained for the SSR markers segregating in the Rps6 ⫻ rps6 population. Map distance

SSR locus

cM† –

Satt199

35.5

Satt472

2.1

Satt191

Plant Rps genotype Rps6Rps6 rps6rps6 Mean Rps6Rps6 rps6rps6 Mean Rps6Rps6 rps6rps6 Mean


AB

BB

7 5

no 8 20

4 7

12 2

7 12

0 18

12 1

7 13

0 18

Recombination P ⫾ SE 0.42 0.47 0.45 0.18 0.25 0.23 0.18 0.25 0.22

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.16 0.12 0.10 0.13 0.10 0.08 0.13 0.10 0.08

† Linkage Group G map distance between the indicated SSR and the SSR immediately above (from Cregan et al., 1999). Other Linkage Group G SSRs were not parentally polymorphic in this population.

1226


more tightly linked to Rps4 (Table 8) than it was to Rps6 (Table 9). Satt012, Satt288, and Sct_187, which flank Satt191 or Satt472, were not polymorphic in this Rps6 population. Satt199, located farther above these markers, was parentally polymorphic, but was not linked to Rps6 (Table 9).

DISCUSSION A major reason for identifying linkage between molecular markers and genes encoding plant resistance to pathogens is to identify molecular “tags” for MAS. Simple sequence repeats (SSRs) are PCR-mediated, singlelocus, codominant, multiple-allele markers that only require small amounts of sample DNA for detection. When used with convenient, inexpensive agarose gels and non-radioactive visualization techniques, they become very “breeder friendly” for MAS applications. The recent integration of 663 SSRs into the public genetic map of the soybean (Cregan et al., 1999) represents a significant upgrade from the previous soybean map based on RFLP markers (Shoemaker and Specht 1995) in terms of cost, convenience, and specific locus identity. More mapped SSRs, which will bring the total number of SSRs to more than 1000, will be released to the public soon. Tight linkages of SSRs with the various Rps loci in soybean would certainly be useful to breeders and pathologists interested in using MAS to mitigate the impact of Phytophthora root rot on this crop. The goal of this research was to identify SSR markers linked to the Rps1 to Rps6 loci. Linkages were found for all but Rps5, but only the linkages (all P ⬍ 0.05) of Satt584, Satt530, Satt152, and Satt159 with the Rps1 locus, and the linkage (P ⬍ 0.09) of Satt472 with the Rps4 locus, were close enough to be possibly useful in MAS applications. Linkages of p ⫽ 0.05 or less are generally required for efficient MAS, although this restriction can be relaxed if two markers flanking the locus of interest are available (Tanksley, 1983). Low SSR polymorphism between the Harosoy and NIL parents precluded the identification of exceptionally tight linkages for each Rps locus (see later discussion). However, the SSR–Rps linkages that were detected in this study were useful in other ways. First, we directly confirmed the SSR map position of those Rps loci whose map positions on the soybean SSR map were heretofore inferred on the basis of map positions of multi-locus RFLP markers linked to those Rps loci (Diers et al, 1992; Cregan et al., 1999). With this study we have established that the Rps1, Rps2, Rps3, and Rps4 loci clearly belong to the SSR linkage groups N, J, F, & G, respectively. Second, we were able to identify a LG-G map position for Rps6, the only Rps locus not yet positioned on the SSR map. Our linkages of SSRs with Rps4 and Rps6, and the linkage of T005 to both Rps4 and Rps5 (Diers et al., 1992), suggests that these three loci may all be linked on LG-G. The literature is not clear as to whether complementation tests were ever performed to determine if Rps4, Rps5, and Rps6 are an allelic series at one monogenic locus (like the multi-allelic Rps1 locus), or are a cluster of closely linked, but recombinably sepa-

rate loci (Athow et al., 1980; Buzzell and Anderson, 1981; Athow and Laviolette, 1982; Diers et al., 1992). Both kinds are evident in other species. For example, the L gene conferring rust resistance in flax consists of at least 13 different alleles at one locus that differ by point mutations as well as major structural changes (Ellis et al., 1999). In contrast, at least 10 separate DM genes, mapping to a single cluster, confer resistance to downy mildew in lettuce (Meyers et al., 1998), and in barley, the Mla locus for powdery mildew resistance consists of a cluster of several linked genes and at least 11 Resistance Gene Analogs (Wei et al, 1999). The soybean genomic segment containing the Rps4, Rps5, & Rps6 genes would seem to be worthy of further research in that regard. The ideal MAS scenario would be one in which each Rps allele for resistance is completely linked to an SSR allele not present in the elite germplasm pool, so that MAS would be applicable to any mating of the donor parent to a recurrent (elite) parent. Moreover, for every Rps locus with multiple alleles, each of its alleles would ideally be linked to a different SSR allele, thereby allowing the identification, in resistant x resistant matings, of those progeny possessing each desired resistance allele. The opportunity for both of the above scenarios exists. Narvel et al. (2000), in a survey of 40 plant introductions (PIs) and 39 elite lines with 74 SSRs, observed a total of 397 different SSR alleles, of which 138 were detected in the PIs only (i.e., not present in the elite lines). More than 85% of the PI-specific 138 alleles had a frequency of 0.25 or less. Two obstacles were encountered in our search for such SSR–Rps linkages. First, there was often a paucity of markers in the genomic regions containing resistance alleles. For example, SSR markers were scarce in the LG-J region containing Rps2, and in the LG-G region containing Rps4, Rps5, and Rps6 (Cregan et al., 1999). This situation is improving as more SSR markers are currently being added to the SSR-sparse regions of the soybean genetic map (Cregan, 2001, unpublished data). Second, when SSRs were abundant in a genomic region, parental SSR polymorphism was often low. This was the case for the LG-F region containing Rps3 (Cregan et al., 1999), where only four of the many available SSRs were polymorphic in our Rps3 population. The only practical solution to this parental monomorphism problem is to sample more parents by creating more than one cross segregating for a given resistance gene. The SSR genotyping of just those F2 individuals homozygous recessive for a gene of interest proved to be a convenient and efficient means of confirming (or refuting) the tightest putative linkages, which were the ones of interest. Since only the homozygous recessives of an F2 population undergo SSR genotyping, the cost of genotyping is but a quarter of that needed to genotype the entire F2 population. Thus, for approximately the same cost of creating one segregating population in which all 100 F2 individuals were SSR genotyped, one could create four different segregating populations of 100 F2s each (from mating one donor Rps parent to four divergent rps parents) and SSR genotype only the four


sets of approximately 25 homozygous recessives. In this way, the probability of encountering parental polymorphism in the genomic region of interest would be greater. The trade-off is that the 100 F2 individuals in each of four populations would still have to be phenotyped to identify the 25 homozygous recessives. Finally, we would recommend SSR genotyping of the F2 homozygous recessive class to soybean geneticists who, on the basis of a classical inheritance study, have identified a new classical gene for a qualitatively scored trait. If the leaf tissue is not available, then one could save seed of the parents and seed of (at least) 25 of the F2 or F3 homozygous recessives. SSR genotyping could be performed on these homozygous recessives, which could lead to the positioning of the new gene on the SSR map. Putting such genes on the map as they are discovered might be of interest to geneticists wishing to clone particular genes of interest. Moreover, when genes affecting the same trait are known to exist, the Soybean Genetics Committee requires allelism tests before the approval of a symbol for a new gene is granted. Comparison of the map position of a new gene with the map positions of existing genes would help the geneticist to determine which of the many possible complementation tests are truly necessary. REFERENCES Allard, R.W. 1956. Formulas and tables to facilitate the calculation of recombination values in heredity. Hilgardia 24:235–278. Akkaya, M.A., A. Bhagwat, and P.B. Cregan. 1992. Length polymorphisms of simple sequence repeat DNA in soybean. Genetics 132: 131–1139. Anderson, T.R., and R.I. Buzzell. 1992. Inheritance and linkage of the Rps7 gene for resistance to Phytophthora rot of soybean. Plant Dis. 76:958–959. Athow, K.L. 1987. Fungal diseases. p. 689–727. In J.R. Wilcox (ed.) Soybeans: Improvement, production, and uses. 2nd ed. Agron. Monogr. 16. ASA, CSSA, and SSSA, Madison, WI. Athow, K.L., and F.A. Laviolette. 1982. Rps6, a major gene for resistance to Phytophthora megasperma f. sp. glycinea in soybean. Phytopathology 72:1564–1567. Athow, K.L., F.A. Laviolette, E.H. Mueller, and J.R Wilcox. 1980. A new major gene for resistance to Phytophthora megasperma var. sojae in soybean. Phytopathology 70:977–980. Bernard, R.L., P.E. Smith, M.J. Kaufmann, and A.F. Schmitthenner. 1957. Inheritance of resistance to phytophthora root and stem rot in the soybean. Agron. J. 49:391. Botstein, D., R.L. White, M. Skolnick, and R.W. Davis. 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32:314–331. Buzzell, R.I., and T.R. Anderson. 1981. Another major gene for resistance to Phytophthora megasperma var. sojae in soybean. Soybean Genet. Newsl. 18:30–33. Cregan, P.B., T. Jarvik, A.L. Bush, R.C. Shoemaker, K.G. Lark, A.L. Kahler, T.T. Van Toai, D.G. Lohnes, J. Chung, and J.E. Specht. 1999. An integrated genetic linkage map of the soybean genome. Crop Sci. 39:1464–1490. Diers, B.W., L. Mansur, J. Imsande, and R.C. Shoemaker. 1992. Map-

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