Novos genes de resistência à ferrugem-asiática-da-soja: teste de alelismo para os locos Rpp2 e Rpp4

New genes conferring resistance to Asian soybean rust: allelic testing for the Rpp2 and Rpp4 loci

GENÉTICA

New genes conferring resistance to Asian soybean rust: allelic testing for the Rpp2 and Rpp4 loci

Novos genes de resistência à ferrugem-asiática-da-soja: teste de alelismo para os locos Rpp2 e Rpp4

Larissa Di Cássia Laperuta^I; Carlos Alberto Arrabal Arias^II; Aliny Simony Ribeiro^II; Breno Francovig Rachid^I; Pedro Henrique Braga Pierozzi^I; José Francisco Ferraz de Toledo^II; Antonio Eduardo Pípolo^II; Geraldo Estevam de Souza Carneiro^II

^IUniversidade Estadual de Londrina, Departamento de Biologia Geral, Centro de Ciências Biológicas, Caixa Postal 6001, CEP 86051-990 Londrina, PR, Brazil. E-mail: [e-mail], [e-mail], [e-mail]

^IIEmbrapa Soja, Caixa Postal 231, CEP 86001-970 Londrina, PR, Brazil. E-mail: [e-mail], [e-mail], [e-mail], [e-mail], [e-mail]

ABSTRACT

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 significant 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 2.5x10⁴ 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.

Index terms:Glycine max, Phakopsora pachyrhizi, inheritance, vertical resistance.

RESUMO

O objetivo deste estudo foi desenvolver testes de alelismo, inclusive cruzamentos entre um grupo de genótipos resistentes à ferrugem, do banco de germoplasma de soja da Embrapa, e as duas fontes de resistência com os genes Rpp2 e Rpp4. A ferrugem-asiática-da-soja, causada pelo fungo Phakopsora pachyrhizi, tem gerado perdas significativas na produtividade da cultura e preocupado os agricultores brasileiros. Até recentemente, existiam quatro genes de resistência descritos na literatura, mas somente Rpp2 e Rpp4 continuam efetivos no Brasil. Vinte e seis fontes com resistência a P. pachyrhizi foram cruzadas com PI 230970 e PI 459025 (fontes dos genes Rpp2 e Rpp4, respectivamente), e as plantas da geração F₂ foram infectadas com uma suspensão com 2,5x10⁴ esporos por mililitro e analisadas em casa de vegetação após 20 dias, para verificação da presença de lesões de resistência (RB) ou de suscetibilidade (TAN). Foram aplicados testes de qui-quadrado para investigar as hipóteses de segregação independente ou de alelos de resistência nos locos Rpp2 e Rpp4. Genes de resistência provenientes de PI 197182, PI 230971 e PI 417125 não segregaram em cruzamentos com a PI 230970, o que indica que esses genótipos apresentam um único gene de resistência presente no loco Rpp2. Cruzamentos com os outros 23 genótipos resultaram em populações segregantes, o que indica que estas possuem genes que não pertencem aos locos Rpp2 e Rpp4.

Termos para indexação:Glycine max, Phakopsora pachyrhizi, herança, resistência vertical.

Introduction

The development of cultivars resistant to Asian soybean rust (ASR) - caused by the Phakopsora pachyrhizi fungus - became the first priority of Brazilian soybean breeding programs since 2001, when the disease was first detected in Southern Brazil. Four dominant genes were described as sources of resistance - Rpp1, Rpp2, Rpp3 and Rpp4 - identified among plant introductions (PIs) and cultivars (Bromfield & Hartwig, 1980; McLean & Byth, 1980; Bromfield & Melching, 1982; Hartwig, 1986). McLean & Byth (1980) studied the inheritance of rust resistance in PI 200492, Tainung 3 and Tainung 4, using the Australian Q-1 isolate, and concluded that resistance is dominant over susceptibility. Working with another Australian isolate (Q-2), the same authors observed that PI 200492 and Tainung 3 were susceptible, while Tainung 4 remained resistant and, therefore, they should carry different resistance genes. The authors named Rpp1 the dominant resistance gene from PI 200492 (McLean & Byth, 1976).

Bromfield & Hartwig (1980) found a single dominant gene determining the resistance of PI 230970 to India-73-1, Philippines-77-1 and Taiwan-72-1 isolates. Hartwig & Bromfield (1983) studied the relationship among the resistance of PI 200492, PI 230970 and PI 462312, using India-73-1 and Taiwan-72-1 isolates. They named Rpp2 and Rpp3 the resistance genes derived from PI 230970 and PI 462312, respectively. Another isolate of the fungus, Taiwan 80-2, broke the resistance of PI 200492, PI 230970 and PI 462312. The genotype PI 459025 was identified as resistant to Taiwan-80-2 and also to India-73-1 and Taiwan-72-1 isolates. Hartwig (1986) crossed the four PIs and concluded that PI 459025 carries a single resistance gene in a different locus (gene Rpp4, in the Rpp4 locus). Still according to Hartwig (1986), Taiwan-72-1 isolate defeated the resistance of PI 200492 and PI 462312, but not those of PI 230970 and PI 459025. Other plant introductions as PI 417125, PI 203398 (Abura), PI 416764, PI 417115, PI 416819, PI 340050 and PI 417503 were studied and presented resistance reaction to P. pachyrhizi (Miles et al., 2006). More recently, other ASR major resistance genes, though recessive, were identified in the advanced Brazilian soybean line BR01-18437 (Pierozzi et al., 2008), and in PI 200456 and PI 224270 (Calvo et al., 2008). Finally, the fifth ASR resistance locus, Rpp5, and a recessive allele from PI 200456 and dominant alleles from PI 200526, PI 200487 and PI 471904 were identified (Garcia et al., 2008).

Historically, the ASR fungus has shown great capacity to develop new races and overcome each resistant gene along time. In Brazil, some genotypes, initially identified 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 efficiency 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.

Materials and Methods

The genetic material selected for allelic testing included 26 PIs or cultivars that were identified as bearers of genes resistant to ASR, under greenhouse conditions at Embrapa Soja, Londrina, PR, Brazil: PI 197182, PI 230971, PI 417125, GC 84058-21-4, PI 408251, PI 379618 TC1, Nova Santa Rosa, PI 203398 (Abura), PI 423966, PI 416764, PI 417115, GC 84051-9-1, PI 398526, PI 416819, PI 339866, PI 340050, PI 417503, PI 417421, PI 203406, FT 87-17893, PI 417074, PI 408205, GC 84058-18-4, PI 416810, PI 200487 (Kinoshita) and PI 423962 (Hyuuga). A single plant harvested from each of resistant materials was used in crosses with the two testers PI 230970 and PI 459025 bearing the resistant genes Rpp2 and Rpp4, respectively. Parents, their F₁ generation and the experimental materials, which included parents and their respective biparental cross derived F₂ generations in a total of approximately 4,730 individuals, were grown in a greenhouse at Embrapa Soybean, Londrina, PR, Brazil. Morphologic traits such as flower, pubescence and hilum color were used to eliminate selfed parent individuals from the F₁ and F₂ generations.

Two experiments were carried out to evaluate the segregation pattern of the F₂ generations. The first one was performed with included six individuals of each of the 21 parents and 60 to 140 individuals of the F₂ generations, and was sown on October 15, 2004. Plants were grown in greenhouse in randomly distributed pots each one containing five to seven plants from the same cross. Plants were individually scored. The second experiment was installed to confirm 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 F₂ 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 efficiency. 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 filter 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 2.5x10⁴ 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 fine 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 classification of their reaction types as reddish-brown (RB - indicating resistance) and light-brown or "tanish" (TAN - indicating susceptibility). The RB lesions are dark with little sporulation, while TAN lesions are lighter with abundant sporulation.

Chi-square tests (χ²) 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 F₂ generation. Only those crosses showing at least 60 F₂ 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 identified.

Table 1 shows the segregation pattern obtained in the F₂ 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 F₂ seeds produced and on the difficulties to classify plants with few lesions or without lesions. Therefore, only those individual F₂ plants expressing typical TAN or RB reaction were considered for analyses.

The investigated resistant sources were divided in five groups: I, F₂ 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, F₂ generations that segregated in a 15:1 ratio for both Rpp2 and Rpp4 loci; III, F₂ generations that segregated in a 13:3 ratio for both Rpp2 and Rpp4 loci; IV, F₂ generations that segregated in a 13:3 ratio for the Rpp2 locus and in a 15:1 ratio for the Rpp4 locus; V, F₂ 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 classified 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 F₂ 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 classified 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 first reported in the soybean line BR01-18437 (Pierozzi et al., 2008), and then in PI 200456 and PI 224270 (Calvo et al., 2008). The existence of recessive ASR-resistance genes will increase the breeders attention to the possibility of obtaining F₁ plants with TAN reaction that should not be discarded.

The segregation pattern obtained in the F₂ 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), confirming 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 confirms the results obtained from the cross with the gene Rpp2 (15RB:1TAN) but not with the gene Rpp4 (13RB:3TAN).

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), confirming 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 identification 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 (χ² 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 difficulties 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 classified in Table 2 as new genes can be different alleles in the Rpp1 or Rpp3 loci or, alternatively, genes derived of nondescribed Rpp loci.

Conclusions

1. A group of 23 soybean rust-resistant sources - GC 84058-21-4, PI 408251, PI 379618 TC1, Nova Santa Rosa, PI 203398 (Abura), PI 423966, PI 416764, PI 417115, PI 416819, GC 84058-18-4, PI 398526, 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) - carry resistance genes that are not in the Rpp2 or in the Rpp4 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.

Acknowledgements

To Embrapa Soja and to Conselho Nacional de Desenvolvimento Científico e Tecnológico for financial support.

Received on August 7, 2008 and accepted on November 19, 2008

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Datas de Publicação

• Publicação nesta coleção
  12 Jan 2009
• Data do Fascículo
  Dez 2008

Histórico

• Aceito
  19 Nov 2008
• Recebido
  07 Ago 2008