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Two Major Recessive Soybean Genes Conferring Soybean Rust Resistance

^a Tropical Melhoramento e Genética Ltda., Rodovia Celso Garcia Cid, Km 87, CEP 86183-600, Cambé, PR, Brazil
^b Fundação de Apoio a Pesquisa Agropecuária do Estado de Mato Grosso–Fundação MT, Av. Antonio Teixeira dos Santos, 1559, CEP 78750-000, Rondonópolis, MT, Brazil

^* Corresponding author (ebersoncalvo{at}tmg.agr.br).

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Soybean rust (SBR) caused by Phakopsora pachyrhizi Syd. is currently the most threatening fungal disease of soybean [Glycine max (L.) Merr.] in the Americas. Development of resistant or tolerant cultivars is a major goal in several soybean breeding programs. Four loci, all carrying dominant alleles that confer a resistant phenotype, have been described. We investigated the genetic basis of the resistance in PI 200456 and PI 224270 by crossing each of them with a susceptible cultivar (CD 208). Phenotypic segregation ratios for F₂ plants and F_2:3 lines showed that the resistance in each resistant parent was controlled by a single recessive gene. A test for allelism demonstrated that these genes are non-allelic. This is the first report of recessive genes controlling SBR resistance in soybean and may represent a different type of resistance for breeding programs aimed at development of more durable resistance.

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS AND DISCUSSION REFERENCES

 
SOYBEAN RUST (SBR) is a very aggressive soybean disease caused by the fungus Phakopsora pachyrhizi Syd. It was first described in Japan over 100 years ago (Bromfield and Hartwig, 1980) and has been reported to damage soybean in several countries in Asia, Australia, and Africa (Kochman, 1977; Sharma and Mehta, 1996; Akinsanmi et al., 2001). Recently, the disease has received considerable attention from soybean growers and researchers after it was first reported in South America in 2001 (Paiva and Yorinori, 2002) and in North America in 2004 (Schneider et al., 2005). Crop damage potential is very high given that these two continents account for more than 70% of the world soybean production and the fact that the disease can cause yield losses that vary between 20 and 80% (Yorinori et al., 2005).

In Brazil, the disease was first reported in the southern regions in 2001 and rapidly moved to central Brazil in the following year (Yorinori et al., 2005). Today it is found in all soybean production areas of the country and is controlled mainly through the application of triazol- and strobirulin-based fungicides. Use of fungicides in soybean production in Brazil has increased from approximately $100 million (U.S.) to almost $800 million since the appearance of SBR (Sindag, unpublished data and under consult rights of the participating companies, 2006). Due to its aggressiveness and the difficulties in detection of the fungus early in the epidemic cycle, farmers have a very short time-frame to apply fungicides for control of the disease. Therefore, even with fungicide applications, yield losses are frequently associated with SBR occurrence. Latest estimates suggest that SBR has caused over $2.2 billion economical losses during the 2006–2007 growing season in Brazil (Embrapa Soja, 2007). In addition, there could be adverse environmental impact associated with the increase in fungicide use.

One approach to reduce the use of fungicide for managing SBR is to breed soybean cultivars with tolerance or resistance to the fungus. Development of such cultivars is enhanced by the understanding of the genetic basis of the resistance or tolerance. Although the screening of tolerant lines has been pursued (Shin and Tschanz, 1986; Hartman, 1996), there are no reports of the genetic basis of the tolerance in the literature. On the other hand, resistant Glycine max germplasm have been found in several countries and the genetic basis of the resistance has been reported. Four SBR-resistance loci (Rpp1, Rpp2, Rpp3, Rpp4) have been described (McLean and Byth, 1980; Bromfield and Hartwig, 1980; Bromfield and Melching, 1982; Hartwig, 1986) and for each of them a dominant allele causes resistance. Moreover, investigations in other pathosystems have shown that single R-gene mediated resistance can also be a key component even in polygenic, quantitative disease tolerance (Li et al., 2001).

As part of an ongoing breeding program we have been screening germplasm for SBR resistance and tolerance. We identified PI 200456 and PI 224270 as possessing resistance to SBR. Our objective was to determine the genetic basis of the SBR resistance in PI 200456 and PI 224270.

	MATERIAL AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Plant Material
As a result of a routine screening procedure in Tropical Melhoramento e Genética Ltda.'s (TMG, Cambé-PR, Brazil) soybean exotic germplasm collection, several accessions showing either resistance or tolerance to soybean rust were identified. Two accessions from Japan, PI 200456 and PI 224270 (USDA-ARS germplasm collection), were identified as resistant according to our screening procedures (see below) and were crossed to the susceptible Brazilian cultivar CD 208 (Coodetec, Cooperativa Central de Pesquisa Agrícola, Cascavel-PR, Brazil). The F₁ seeds were obtained and two seeds of each cross combination were grown to produce the F₂ seeds.

A total of 204 (102 from each F₁ plant) F₂ seeds were sown in 8 L plastic pots filled with a mixture of soil, manure, and sand (5:3:2 v/v/v) in the greenhouse. Seeds from both parents (eight seeds) as well as remnant F₁ seeds (10 seeds) were also sown. F₂ plants were evaluated for SBR resistance (as described below) and allowed to set seeds. Plants were individually threshed to obtain the F_2:3 progeny. Twenty seeds from each F_2:3 line and the parents were sown in the greenhouse and evaluated for soybean rust resistance following the same procedure used for F₂ plants.

Disease Resistance Screening
The soybean rust isolate used was obtained by collecting spores from naturally infected greenhouse plants on the susceptible cultivar BRSMS Bacuri (Embrapa, Empresa Brasileira de Pesquisa Agropecuária, Londrina-PR, Brazil) in Cambé-PR, Brazil, during the summer of 2004. This cultivar carries a rust-resistance gene from cultivar FT-2 that had its resistance broken during the summer of 2003, shortly after the first appearance of the disease in Brazil. It seems that this isolate is very representative. We have been field-testing different resistant PIs and breeding lines in other parts of Brazil and until now we have not detected distinct behavior of these genotypes.

Samples of the collected isolate were increased on artificial infection of the susceptible cultivar BRS 154 in the greenhouse and were used either for screening purposes or frozen in liquid N₂ for future use. Samples of this isolate are available on request.

The SBR resistance screening was performed by applying the P. pachyrhizi inoculum as a water suspension containing 50 x 10³ spores per mL supplemented with 0.02% (v/v) of Tween 20. Spores were collected at the day of inoculation from the susceptible cultivar BRS 154 (Embrapa, Empresa Brasileira de Pesquisa Agropecuária, Londrina-PR, Brazil). The first inoculation was applied at the V2 stage (Fehr and Caviness, 1977) followed by two additional inoculations 6 and 12 d later. Ten days after the final inoculation, plants were individually scored as susceptible (S) or resistant (R). Plants rated as susceptible developed the ‘tanish’ (TAN) type of lesions followed by sporulation, while the resistant plants showed the typical dark ‘reddish-brown’ (RB) lesion (Bromfield, 1984). Phenotypic (F₂) and "genotypic" (F_2:3) segregation hypothesis were tested with a Chi-Square (²) test.

Allelism test
An allelism test was conduct by crossing PI 200456 and PI 224270. The F₁ seeds were planted and the resulting F₂ seeds were harvested. A total of 72 F₂ seeds were planted and the F₂ plants were screened for soybean rust resistance as described above.

	RESULTS AND DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Since the soybean rust isolate used did not originate from a single lesion, it is possible that it represents a mixture of more than one race. Therefore, all multiplications of the isolate (in BRS 154), as well as screening procedures, were monitored for the appearance of mixed (TAN and RB) lesion types. Currently, no mixed lesion types have been observed in our greenhouse inoculations. Furthermore, the fact that the parents were grown along with the segregating populations during the screening procedures indicated a consistent parental response during the experiments.

The resistance reaction was determined by the presence of RB lesions. In the F₂ generation 53 out of 201 F₂ plants derived from PI 200456 x CD 208 population were resistant to SBR (Table 1 ). Similarly, 43 of 195 plants from the F₂ PI 224270 x CD 208 population developed the resistant RB lesion. The phenotypic segregation of the SBR reaction in both F₂ populations fit a 1 resistant plant to 3 susceptible plant ratio (Table 1), supporting the hypothesis that a recessive gene controls the resistance phenotype. This hypothesis was confirmed by the 1 (homozygous susceptible):2 (segregating):1 (homozygous resistant) line segregation obtained in the F_2:3 progeny test of both populations (Table 2 ).

Due to the economic importance of soybean, several disease resistance genes have been identified in the species (Palmer et al., 2004). Out of 45 loci encoding disease resistance genes for which gene symbols have been assigned, in only six of them, Rxp (Xanthomonas campestris pv. Glycines), Rpv2 (peanut mottle virus), Rhg1, Rhg2, Rhg3 (cyst nematode– Heterodera glycines), and Rrn (reniform nematode–Rotylenchulus reniformis), the resistance is encoded by a recessive allele (Palmer et al., 2004). Although rpv2 and rrn have not been largely used, rxp and rhg1–3 alleles have been widely employed in breeding programs around the world and have remained effective against its pathogens for more than 40 years.

Molecular cloning and characterization of more than 20 dominant disease-resistance genes (R genes) suggest that they encode proteins that act as receptors in triggering the disease resistance response in the cells (McDowell and Simon, 2006). On the other hand, only three recessive genes have been cloned: mlo (Büschges et al., 1997), edr-1 (Frye et al., 2001), and RRS1-R (Deslandes et al., 2002). They all seem to encode proteins with functional domains different from the common NBS-LRR domain present in almost all dominant R genes (Chu et al., 2004). Therefore, the recessive genes presented here may help in the understanding of the function of recessive genes in disease resistance.

Because of the recessive nature of the resistance in PI 200456 and PI 224270, we investigated whether they were allelic. The possible ratios to confirm this hypothesis in the F₂ population would be either 9 susceptible:7 resistant, or 15 susceptible:1 resistant (if we consider an epistatic effect). Phenotypic segregation was observed in the resulting F₂ population, as expected for two alleles at different loci (Table 3 ). However, the segregation data pointed to a 3:1 (resistant:susceptible) ratio, indicating that a single dominant gene is determining the resistant in this PI x PI cross. This hypothesis was discarded because the recessive nature of the genes present in both PI's was clearly demonstrated (see Table 1 and Table 2) using both F₂ and F_2:3 populations. Moreover, molecular mapping of these genes also indicate that they are located at different loci (Garcia et al., 2008). Apparently there is an uncommon gene action involved in determining resistance when these two recessive genes are combined. This hypothesis should be further clarified with more detailed studies that we are conducting. It also will be interesting to verify if these genes are allelic to any of the four (Rpp1, Rpp2, Rpp3, Rpp4) resistant dominant genes previously reported or if they reside at a new locus.

Wheat research has also shown that the genetic background may determine the type of gene action in leaf rust resistance genes (Dyck and Samborski, 1974). This does not appear to be the case with the Rpp alleles reported in this study since we have been using both PI 200456 and PI 224270 as parents in our soybean breeding program and their segregation is always consistent with a recessive gene conditioning resistance (data not shown).

One of the key issues in the development of resistant cultivars is the possibility of the pathogen to overcome the host resistance. In fact, several races of P. pachyrhizi have been described (Yamaoka et al., 2002). SBR was first reported in Brazil over-wintering on soybean plants during May 2001 (Paiva and Yorinori, 2002). In the 2001–2002 growing season a couple of resistant commercial cultivars were found. Preliminary analysis of race specificity at that time using the differential soybean genotypes indicated that all previously reported G. max Rpp genes (Rpp1, Rpp2, Rpp3, Rpp4) conferred resistance to that soybean rust population (Yorinori et al., 2005). However, in the following growing season (2002–2003) a new rust population that defeated the Rpp1 and Rpp3 resistance appeared in Central Brazil. Apparently this population is now prevalent since we have not been able to detect RB lesions on genotypes that carry the Rpp1 or Rpp3 allele in our recent screenings.

Use of soybean rust isolates obtained from single lesions should reveal if in fact a mixture of races occurs in Brazil, especially in the inoculum used during this study. Nevertheless, the fact that a monogenic segregation to the resistance and no mixed (RB/TAN) lesion types were observed in homozygous resistant plants suggests that, if the inoculum is comprised of a mixture of races, these races are very similar since they are behaving identically on the genotypes used in the present study. Also, it is important to note that because we have a nation-wide breeding program in Brazil, we have been field-testing these PIs along with derived breeding lines in other parts of Brazil, especially in Central Brazil. Up to now we have not detected different behavior of these genotypes in these different regions.

It seems that the SBR fungus has an ability to accumulate genes for virulence (Bonde et al., 2006). It will be interesting to compare the response of plants homozygous for the recessive and dominant resistance-genes across a diversity of isolates obtained in Brazil and in countries where the disease has been present for a long period, such as Japan, Australia, and China. Results from this comparison could reveal whether the recessive alleles reported in this paper may represent a more durable source of resistance than the currently available Rpp genes. At least in some pathosystems the recessive R-genes appear to confer durable broad-spectrum disease resistance (Deslandes et al., 2002). In any case, the new genes described here should contribute to the development of soybean rust-resistant soybean cultivars.

	ACKNOWLEDGMENTS

 
We would like to thank Marcelo Soares Baço for his technical assistance in the greenhouse screenings. This project has received the financial support from Fundação de Amparo à Pesquisa do Estado de Mato Grosso–FAPEMAT and from Financiadora de Estudos e Projetos–FINEP/MCT (Ação Estruturante Transversal).

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIAL AND METHODS RESULTS AND DISCUSSION REFERENCES

 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Received for publication February 25, 2008.

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