New genes conferring resistance to Asian soybean rust : allelic testing for the Rpp 2 and Rpp 4 loci

The objective of this study was to conduct allelic tests including crosses between a group of rust resistant genotypes from Embrapa’s soybean germplasm collection and PI 230970 and PI 459025, which carry the Rpp2 and Rpp4 genes, respectively. Asian Soybean rust (ASR) caused by the fungus Phakopsora pachyrhizi has resulted in signifi cant yield losses and concern among Brazilian farmers. Until recently, there were four resistance genes (Rpp1 through Rpp4) described in the literature, but only Rpp2 and Rpp4 are still effective in Brazil. Twenty-six sources of resistance to P. pachyrhizi were crossed with PI 230970 and PI 459025 (Rpp2 and Rpp4 gene sources, respectively) and plants of their F2 generations were infected with a suspension containing 2.5x10 spores per milliliter and assessed in a greenhouse after 20 days, for the presence of resistant (RB) or susceptible (TAN) lesions. Chi-square tests were applied to investigate the hypotheses of independent or allelic resistance gene segregations. ASR resistant genes derived from PI 197182, PI 230971 and PI 417125 did not segregate in crosses with PI 230970, which indicates that these genotypes have a single resistance gene in the Rpp2 locus. Crosses with the other 23 genotypes resulted in segregating populations, suggesting that their resistance genes do not belong to Rpp2 or Rpp4 loci.

Historically, the ASR fungus has shown great capacity to develop new races and overcome each resistant gene along time.In Brazil, some genotypes, initially identifi ed as resistant, had their resistance defeated by an isolate from Mato Grosso State (MT) (Yorinori et al., 2004), which also defeated the resistance provided by Rpp1 and Rpp3 genes.Recent tests involving soybean accessions from Brazil and USA germplasm banks indicated that there are several plant introductions expressing resistance to P. pachyrhizi.There is, however, little information regarding whether this resistance is conferred by genes different from those Rpp1-Rpp4 already described.Knowledge about the genetic control of ASR resistance in these new available sources can increase the development effi ciency of resistant soybean varieties and contribute to the disease management worldwide.
The objective of this work was to conduct allelic tests by crossing a group of 26 ASR resistant sources with the two remaining effective resistance Rpp2 (PI 230970) and Rpp4 (PI 459025) genes.
Two experiments were carried out to evaluate the segregation pattern of the F 2 generations.The fi rst one was performed with included six individuals of each of the 21 parents and 60 to 140 individuals of the F 2 generations, and was sown on October 15, 2004.Plants were grown in greenhouse in randomly distributed pots each one containing fi ve to seven plants from the same cross.Plants were individually scored.The second experiment was installed to confi rm the segregation pattern obtained for some of the resistance sources: PI 230971, PI 416819, PI 200487 (Kinoshita), GC 84058-18-4 and GC 84051-9-1, on Feb 01, 2005.Three plants of each parent and 70 to 100 F 2 plants from each cross were grown in pots randomly distributed in the greenhouse.Twenty-four plants of the susceptible cultivar BRSMS Bacuri were also sown in each experiment, to check the inoculation effi ciency.Each pot contained three plants that were individually scored.
The P. pachyrhizi isolate derived from MT isolate (originated in Mato Grosso State), and which defeated the resistance expressed by the Rpp1 and Rpp3 genes, was used for the inoculations.Based on ITS sequence, this isolate is probably related to the MUT Zimbabwe isolate (GenBank accession no.AF333499), showing an identity of 99.8% (Silva et al., 2008).The MT isolate was constantly inoculated, at greenhouse conditions, on BRSMS Bacuri soybean cultivar, which served as fi lter against the Southern Brazilian isolate to which it is resistant.Spores were collected from leaves showing profuse sporulation, by using a soft brush, and then were placed in distilled water.Since the spores were not collected from a single lesion, they may represent a mixture of multiple races.However, the presence of RB and TAN reactions on the same leave was not observed in our assessments.Inoculum preparation involved a Neubauer chamber to obtain 2.5x10 4 spores per milliliter solution to which Tween 20 was added.Thirteen days after the emergence, the inoculum was sprayed only once on plants, with an atomizer, after 18h, to ensure longer leaf moisture permanence and to avoid deleterious sun effects on the germinating spores.During the night following spraying, the plants were submitted to a fi ne water spray during 15 s every 3 hours.Evaluation was carried out between 20 to 27 days after inoculation (between V6 and V9 soybean growth stage), the observation of all leaves of the plants and classifi cation of their reaction types as reddish-brown (RB -indicating resistance) and lightbrown or "tanish" (TAN -indicating susceptibility).The RB lesions are dark with little sporulation, while TAN lesions are lighter with abundant sporulation.
Chi-square tests (χ 2 ) to check the expected 3:1, 15:1, 13:3, 63:1 theoretical ratios for one, two or three independent genes segregating were applied to the F 2 generation.Only those crosses showing at least 60 F 2 individuals and clear reaction type were considered in the analyses.The parental and BRSMT Bacuri cultivar reactions were used as checks to assess the presence of false resistant scores.

Results and Discussions
The experimental conditions allowed good expression of the genotype reactions to ASR with lesions on leaves becoming visible between the seventh and eighth day after inoculation.The spore mass was visible between the 12 th and the 15 th days after inoculation.The mixed reaction commonly observed on ASR evaluations (Miles et al., 2006), where both RB and TAN lesions appear on the same plant, was not observed in the experiments, including the check plants of susceptible cultivar BRSMS Bacuri.The use of a single isolate as inoculum and the greenhouse environment free of other isolates were the most likely cause of plant homogeneous reactions.
The susceptibility reaction (TAN lesions) of cultivar BRSMS Bacuri and PI 200492 (Rpp1 gene) and PI 462312 (Rpp3 gene) and the resistance reaction (RB lesions) of PI 230970 (Rpp2) and PI 459025 (Rpp4) were consistent with the predominance of the MT isolate in both experiments.According to Hartwig (1986), Taiwan-72-1 P. pachyrhizi isolate broke the resistance of PI 200492 and PI 462312, but not the resistance of PI 230970 and PI 459025.This reaction pattern is similar to that obtained in this study with the Brazilian isolate, suggesting that the Taiwan-72-1 and MT isolates may belong to the same race.Until present studies on ASR races being carried out in Brazil, no results concerning these two isolates are conclusive.Reference should be also made to the study of Silva et al. (2008), based on ITS sequence, which found 99.8% identity between the MT and MUT Zimbabwe isolates (GenBank accession no.AF333499).
The analyses of our data allowed screening of three genotypes with resistant genes belonging to the Rpp2 locus and 23 genotypes with new resistant genes, which showed independent segregation from both the Rpp2 and Rpp4 loci.These two loci remain effective against the MT soybean rust isolate.No new source bearing resistance genes in the Rpp4 locus was identifi ed.
Table 1 shows the segregation pattern obtained in the F 2 populations derived from crosses between each investigated resistance source with PI 230970 and PI 459025.The total number of plants evaluated in each cross depended on the number of F 2 seeds produced and on the diffi culties to classify plants with few lesions or without lesions.Therefore, only those individual F 2 plants expressing typical TAN or RB reaction were considered for analyses.
The investigated resistant sources were divided in fi ve groups: I, F 2 generations that did not show segregation for the Rpp2 locus and segregated in a 15:1 or 13:3 ratio (resistant to susceptible) for the Rpp4 locus; II, F 2 generations that segregated in a 15:1 ratio for both Rpp2 and Rpp4 loci; III, F 2 generations that segregated in a 13:3 ratio for both Rpp2 and Rpp4 loci; IV, F 2 generations that segregated in a 13:3 ratio for the Rpp2 locus and in a 15:1 ratio for the Rpp4 locus; V, F 2 generations that segregated in a 15:1 ratio for the Rpp2 locus and in a 13:3 ratio for the Rpp4 locus.
Genotypes PI 197182, PI 230971 and PI 417125 were classifi ed in group I, thus each one of these carry a single resistance gene located in the same linkage group of the Rpp2 locus already described in the literature.The Rpp2 locus was mapped on soybean linkage group J (Silva et al., 2008) and, with our data, it is not possible to know if the resistant genes derived from those PIs are alleles or if they are in different linked loci.Another resistant gene, though recessive, derived from PI 224270, was also mapped into this same linkage group (Garcia et al., 2008).The difference in dominance mode action from one source to another shows that they may be multiple alleles in the Rpp2 locus.Therefore, breeders trying to pyramiding those genes may not be successful.
Genotypes GC 84058-21-4, PI 408251, PI 379618 TC1, Nova Santa Rosa, PI 203398 (Abura), PI 423966, PI 416764, PI 417115, PI 416819 and GC 84058-18-4 were all included in group II.Therefore, each of these genotypes carries resistance genes at a locus different from Rpp2 and Rpp4.The obtained segregation pattern in the F 2 generations of each cross also supported the hypothesis that each new source carries a single dominant gene controlling resistance.The soybean breeding line BR01-18437, studied by Pierozzi et al. (2008), probably received its resistant gene from PI 203398 (Abura), which was used as high protein genetic source.However, the gene from BR01-18437 is recessive, while the gene from PI 203398 studied here is dominant.Complementary studies are being developed to clarify how dominance of this gene works.
Only one genotype, PI 398526, was classifi ed into group III.The observed segregation pattern, 13 resistant to 3 susceptible, indicated that its resistant gene is recessive or does not express dominance when crossed with PI 230970 and PI 459025.Recessive ASR-resistance genes have been fi rst reported in the soybean line BR01-18437 (Pierozzi et al., 2008), andthen in PI 200456 andPI 224270 (Calvo et al., 2008).
The existence of recessive ASR-resistance genes will increase the breeders attention to the possibility of obtaining F 1 plants with TAN reaction that should not be discarded.
The segregation pattern obtained in the F 2 generation for the resistant sources PI 339866, PI 340050, PI 417503, PI 417421, PI 203406, FT 87-17893, PI 417074, PI 408205, GC 84051-9-1, PI 416810, PI 200487 (Kinoshita) and PI 423962 (Hyuuga) indicated that each one of these genotypes has one gene for resistance to ASR in a locus distinct from Rpp2 and Rpp4.Considering that Rpp2 and Rpp4 genes in the testers are dominant, a 15:1 segregation ratio would be expected, if the resistance genes in the tested genotypes were also dominant, and a 13:3 ratio would be expected if they were recessive.
The results obtained in groups IV and V, however, can only be explained considering a different reaction type of the heterozygous genotype for the resistance locus derived from the tested genotypes, in combination with the homozygous recessive genotype for the Rpp2 and Rpp4 loci.The probability of this genotype combination is 1/8 (or two in each 16 plants).Those genotypes in group IV would produce 13RB to 3TAN and 15RB to 1TAN for the Rpp2 and Rpp4 loci, respectively, while in group V, similar results would be obtained for the Rpp4 and Rpp2 loci, respectively.Therefore, epistatic gene action may have occurred, since the expression of the heterozygous genotype seems to have depended on the tester and, for groups IV or V, i.e. the gene dominance depended on the tester, displaying from complete dominance (15:1) to absence of dominance (13:3).Three types of dominance action were assigned to resistance to ASR, from partial to complete dominant genes (Garcia et al., 2008) and recessive genes (Pierozzi et al., 2008;Calvo et al., 2008).
The ASR-resistance gene from PI 200487 (Kinoshita) source from the previous discussed group was assigned to the locus Rpp5, which was mapped in the linkage group N of soybean (Garcia et al., 2008), confi rming that it is independent of loci Rpp2 and Rpp4 (linkage groups J and G, respectively).Data from Garcia et al. (2008) showed that only one gene with complete dominance confers the resistance of PI 200487, which confi rms the results obtained from the cross with the gene Rpp2 (15RB:1TAN) but not with the gene Rpp4 (13RB:3TAN).Pesq. agropec. bras., Brasília, v.43, n.12, p.1741-1747, dez. 2008 Similarly to PI 200487 (Kinoshita), PI 423962 (Hyuuga) showed ratios 15RB:1TAN and 13RB:3TAN in crosses with Rpp2 and Rpp4, respectively.The ASR-resistance locus of PI 423962 has been mapped on the linkage group C2 (Monteros et al., 2007), confi rming its independence.Another resistance gene derived from the Brazilian cultivar FT-2 had been previously mapped into this same linkage group, but this gene has been already defeated by ASR fungus.
The results of the allelic tests allowed the identifi cation of three investigated sources, which carry genes in the same linkage group where the Rpp2 locus (linkage group J of soybean) is located, and 23 sources with resistance genes mapping out of Rpp2 or Rpp4 loci, which can be used by breeders to develop cultivars resistant to ASR.A summary of these results can be seen in Table 2, which shows the segregation ratios not rejected by chi-square test (χ 2 with p>0.05) for both testers.
At the beginning of this research the objective was to develop allelic tests for all Rpp1 through Rpp4 loci described in the literature.This objective was changed due to diffi culties of maintaining and experimenting with two isolates of the soybean rust fungus and also to the loss of interest of Brazilian breeders in the Rpp1 and Rpp3 genes, which have been defeated by the MT isolate.As consequence, the genes classifi ed in Table 2 as new genes can be different alleles in the Rpp1 or Rpp3 loci or, alternatively, genes derived of nondescribed Rpp loci.
2. Three of the studied resistant sources -PI 197182, PI 230971 and PI 417125 -have at least one allelic or closely linked gene to the Rpp2 locus.

Ratio with Rpp2
Ratio with Rpp4

Table 1 .
Segregation pattern obtained from the F 2 generations derived from crosses between each resistance source with PI 230970 and PI 459025 testers, including the theoretical ratio not rejected by a chi-square test (χ 2 ), the number of evaluated plants (N), lesion type (Lesion), observed (O) and expected (E) number of plants, degrees of freedom (df) and the probability level (p>0.05).

Table 2 .
Summary of the results, showing the segregation ratio obtained for each test cross and the conclusions from allele testings.