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CELL BIOLOGY & MOLECULAR GENETICS

Sequence Characterized Amplified Regions Linked to Rust Resistance Genes in the Common Bean

Departamento de Biologia Geral/BIOAGRO, UFV, 36571-000, Viçosa, MG, Brazil

ebarros{at}mail.ufv.br

	ABSTRACT

TOP ABSTRACT INTRODUCTION Material and methods Results and discussion REFERENCES

 
Uromyces appendiculatus (Pers.) Unger, the causative fungus of rust in common beans (Phaseolus vulgaris L.), consists of many pathotypes or pathogenic races. Cultivar Ouro Negro is resistant to most pathotypes detected in Brazil in the last few decades. We attempted to identify molecular markers linked to a rust resistance gene block present in Ouro Negro. DNA samples extracted from homozygous near isogenic BC₃F_2:3 lines derived from a cross between susceptible cultivar US Pinto 111 and Ouro Negro were grouped, following greenhouse inoculations, into two contrasting bulks, one containing only resistant and the other only susceptible plants. The bulks were amplified with 605 random primers and two of them amplified bands which were heteromorphic between the two bulks. These random amplified polymorphic DNA (RAPD) bands were transformed into sequence characterized amplified regions (SCARs) SCARBA08 and SCARF10. The rust resistance–susceptibility phenotypes and the molecular genotypes with the two SCAR markers of 303 F_2:3 families from US Pinto 111 x Ouro Negro were determined. SCARBA08 and SCARF10 were determined to be 4.3 ± 1.2 and 6.0 ± 1.3 centimorgans (cM) from the rust resistance locus, respectively. These markers are being used in combination with other markers for resistance to rust, anthracnose, and angular leaf spot previously identified in our laboratory to aid the indirect selection of desirable plants in segregating populations in which Ouro Negro has been used as donor parent.

Abbreviations: ASAP, allele-specific associated primers • bp, base pairs • cM, centimorgan • NIL, near isogenic line • RAPD, random amplified polymorphic DNA • SCAR, sequence characterized amplified region

	INTRODUCTION

TOP ABSTRACT INTRODUCTION Material and methods Results and discussion REFERENCES

 
MOST COMMON BEAN cultivars are susceptible to rust, caused by the fungus Uromyces appendiculatus. Field trials have shown losses of up to 68% in the state of Minas Gerais, Brazil (Vieira, 1983) and of about 75% in Puerto Rico (Velez-Martinez et al. 1989) because of U. appendiculatus infection. Several reports indicate that resistance to this disease is controlled by major single dominant genes (Vieira, 1983; Stavely, 1984; Faleiro, 1997). However, there are also reports of resistance determined by a single recessive gene (Zaiter et al., 1989), by two genes (Finke et al., 1986), by two complementary dominant genes (Grafton et al., 1985), and by two independent genes (Grafton et al., 1985). The rust resistance genes identified in common beans have been compiled and a uniform nomenclature has been proposed (Kelly et al., 1996). At least 11 genes have been identified so far (Ur-1 to Ur-11) (Kelly et al., 1996; Stavely, 1998). Most of them confer resistance to multiple races of U. appendiculatus, indicating that they are organized in clusters of race-specific genes.

DNA amplification of near isogenic lines (NILs) (Paran et al., 1991; Martin et al., 1991) or of contrasting DNA bulks from segregating populations (Michelmore et al., 1991) can be used to detect RAPD (Williams et al., 1990; Welsh and McClelland, 1990) markers in close proximity to disease resistance genes. During the development of NILs, these two strategies can be combined by constructing DNA bulks from BC_nF_2:3 individuals from each of two contrasting NILs. This method increases the chance that markers will be identified close to the gene of interest (Michelmore et al., 1991; Haley et al., 1993; Miklas et al., 1993).

Several RAPD markers associated with genes conferring resistance to rust in common bean have been identified (Miklas et al, 1993; Haley et al., 1993.; Johnson et al., 1995). In some cases, the application of these markers has been restricted to the laboratories where they were identified because of reproducibility problems with this technique (Kesseli et al., 1992; Weeden et al., 1992). Sequence characterized amplified regions (Kesseli et al., 1992) and allele-specific associated primers (ASAP) (Weeden et al., 1992) have been proposed as alternative procedures to increase the reproducibility of the RAPD technique. SCARs identify a specific locus by amplification with a defined oligonucleotide primer pair (Paran and Michelmore, 1993). In addition, in many cases these markers can be detected without the need of the gel electrophoresis step by staining the amplification products with ethidium bromide in the amplification reaction tube (Dedryver et al., 1996; Melotto et al., 1996). In the common bean, several SCARs associated with genes for disease resistance have been identified (Adam-Blondon et al., 1994; Melotto et al., 1996).

This work describes the identification of two RAPD markers associated with rust resistance in common beans and their use to develop highly reproducible SCAR markers.

	Material and methods

TOP ABSTRACT INTRODUCTION Material and methods Results and discussion REFERENCES

 
Genetic Material and Biological Assays
Two groups of homozygous BC₃F₂ common bean NILs derived from a cross between resistant cultivar Ouro Negro (donor parent) and susceptible cultivar US Pinto 111 (recurrent parent) and contrasting for rust resistance/susceptibility were used. After the first two backcrosses the resistant plants were fingerprinted by the RAPD technique to select those closer to the recurrent parent. Ouro Negro is a black seeded Mesoamerican cultivar derived from accession Honduras 35. It is resistant to all 13 pathotypes of U. appendiculatus identified in the state of Minas Gerais, Brazil (Faleiro, 1997) and has maintained resistance to all pathotypes for several decades since its introduction into Brazil (Vieira, 1983).

Segregation analyses were performed by the chi-square test and the genetic distances were calculated by the Kosambi function with the aid of the program Mapmaker 3.0, with a LOD score of 3.0 and a maximum recombination fraction of 35% (Lander et al., 1987; Lincoln et al., 1992). For the linkage analyses between the resistance gene block and the molecular markers, a segregating population of 303 F₂ individuals derived from a cross between US Pinto 111 and Ouro Negro was used. The resistant–susceptible phenotypes of the F₂ and BC₃F₂ plants were confirmed by inoculation of 12 individuals derived from each F₂ or BC₃F₂ plant with U. appendiculatus Pathotypes 2, 8, 10, and 11 (Faleiro, 1997) using the multiple inoculation technique (Stavely, 1983). All inoculations were conducted 12 d after sowing with a suspension containing 2 x 10⁴ spores per mL of water and 0.03% (v/v) polysorbate 20. The spores were applied to the first trifoliate leaves with approximately 2/3 of its full development, with the aid of a paint brush.

After inoculation, the plants were kept in a mist chamber (20 ± 1°C and >95% relative humidity) for 48 h under a 12-h photoperiod, and were then transferred to the greenhouse. Twenty days after inoculation disease symptoms were evaluated using the scale proposed by Stavely et al. (1983).

The pathotypes used were collected in the state of Minas Gerais, Brazil, and classified by inoculation of eight differential cultivars (Kentucky Wonder 814, Early Gallatin, 51051, NEP-2, Ecuador 299, Olathe, Mexico 309, and Redlands Pioneer) which have been proposed by Mora-Nuñes et al. (1992) to identify U. appendiculatus pathotypes that occur in Brazil. Spores of these pathotypes are kept in the Fungal Collection of the Biotechnology Center of the Federal University of Viçosa, MG, Brazil, and can be requested from Dr. Everaldo G. de Barros (ebarros@mail.ufv.br).

Identification, Cloning, and Characterization of RAPD Markers
Equimolar amounts of DNA extracted from four BC₃F₂ homozygous resistant and four susceptible plants were used to construct two contrasting DNA bulks. DNA extraction was according to a standard CTAB method (Doyle and Doyle, 1990). The DNA bulks were amplified by the RAPD technique (Williams et al., 1990; Welsh and McClelland, 1990), with 605 random decamer primers (Operon Technologies, Alameda, CA). The reaction volumes, reagent concentrations, and amplification conditions used were described by Corrêa et al. (1999). The amplification products were analyzed on 1.2% (w/v) agarose gel (100 V for 4 h), stained with ethidium bromide (0.2 mg/mL), and visualized under UV light.

Heteromorphic DNA fragments distinguishing the two bulks were tested in the 303 F₂ individuals to determine their distance to the resistance gene block. Those that were close to the gene block (less than 10 cM) were cloned in the vector pGEM-T Easy (Promega, Madison, WI). Competent JM109 Escherichia coli cells were transformed (Ausubel, 1998) and mini-prep DNA from white colonies was sequenced with M13 universal primers flanking the insert (Sanger et al., 1977). On the basis of the sequencing data, two specific SCAR primers were designed and synthesized for each RAPD marker. Synthesis of primers was performed by GIBCO-BRL/Life Technologies (Rockville, MD).

SCAR Analysis
DNA samples extracted from the 303 F₂ individuals in the segregating population were amplified in 25-µL reactions. The same reagents and concentrations were used as in the RAPD reactions, except the primer was replaced by 5 picomoles of pertinent SCAR primer. The thermocycler was programmed for an initial denaturation step of 94°C for 3 min followed by 35 cycles (94°C/15 s, 65°C/1 min, 72°C/1 min 30 s) and one final extension step at 72°C for 7 min. DNA from 303 individuals of the segregating population and the corresponding progenitors were amplified. The amplification products were analyzed as described for the RAPD technique.

	Results and discussion

TOP ABSTRACT INTRODUCTION Material and methods Results and discussion REFERENCES

 
Phenotypic Analyses and Identification of RAPD Markers
The numbers of resistant and susceptible F₂ plants determined with the four U. appendiculatus pathotypes fit a 3:1 ratio (Table 1) . The genotypic ratio based on the symptoms of the corresponding F₃ families fit a 1:2:1 ratio (Table 1), confirming that a single dominant gene, or gene block, confers resistance to rust in cultivar Ouro Negro (Faleiro, 1997).

View larger version (148K): [in this window] [in a new window]   Fig. 1 Electrophoretic analyses of DNA amplification products showing (A) RAPD markers OPF101,072C, (B) SCAR marker SCARF10, (C) RAPD marker OPBA08530C, and (D) SCAR marker SCARBA08. Lanes are as follows: 1, cv. Ouro Negro; 2, cv. US Pinto 111; 3 through 6, F2 resistant plants; and 7 through 10, F2 susceptible plants. M refers to lambda phage DNA digested with EcoRI, HindIII, and BamHI restriction enzymes (size markers)

Development and Analyses of SCAR Markers
Two pairs of primers were designed and synthesized on the basis of the sequences of the cloned RAPD fragments (Michelmore et al., 1991). The sequences of the SCAR primers flanking marker OPF10_1,072C are 5' GGA AGC TTG GTG AGC AAG GA 3' and 5' GGA AGC TTG GCT ATG ATG GT 3' and the corresponding marker amplified was designated SCARF10. The sequences of SCAR primers flanking marker OPBA8_530C are 5' CCA CAG CCG ACG GAG GAG 3' and 5' GCC ATG TTT TTT GTC CCC 3' and the corresponding marker amplified was designated SCARBA8. Figure 1 depicts the amplification patterns showing RAPD and SCAR markers in the progenitors, and F₂ resistant and susceptible plants.

In this work, only two of 605 different 10-mer primers used to amplify the contrasting rust resistant vs. susceptible bulks revealed polymorphisms. In other crosses contrasting for rust resistance involving cultivar Ouro Negro, 800 primers were tested revealing only one polymorphism between the bulks (Ragagnin et al., 1998). The SCAR markers we developed are being now used in our breeding program to characterize and select individuals carrying the Ouro Negro rust resistance gene block. They are highly reproducible and can be used in association with marker OPX11 which is also linked to the resistance gene block present in Ouro Negro (Ragagnin et al., 1998). Those authors obtained a 100% selection efficiency using the SCAR markers associated with the RAPD marker they identified. Our attempts to transform marker OPX11 into a SCAR marker were unsuccessful. DNA from both resistant and susceptible plants was amplified with the designed SCAR primers within a wide range of annealing temperatures (data not shown).Doyle Doyle 1996

	ACKNOWLEDGMENTS

 
This work is part of the PhD Thesis of the first author and was financed by grants from the Brazilian Government: PADCT/FINEP #64.93.0430.00 and FAPEMIG #CBS 1074/96. RXC was supported by a fellowship from CNPq. The authors wish to thank Dr. M. Melotto for her helpful suggestions during the preparation of this manuscript and also Dr. James R. Stavely for providing unpublished data concerning the identity of the resistance gene block present in Ouro Negro.

Received for publication March 3, 1999.

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TOP ABSTRACT INTRODUCTION Material and methods Results and discussion REFERENCES

 

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