RAPD and SCAR markers linked to resistance to frogeye leaf spot in soybean

 

Sebastião Martins Filho¹, Carlos Sigueyuki Sediyama^2,3, Maurilio Alves Moreira^2,4 and Everaldo Gonçalves de Barros^2,5
1Departamento de Engenharia Rural, UFES, Alegre, ES, Brazil.
²Instituto de Biotecnologia Aplicada à Agropecuária (BIOAGRO).
³Departamento de Fitotecnia, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
⁴Departamento de Bioquímica e Biologia Molecular, Universidade Federal de Viçosa, Viçosa, MG, Brazil.
⁵Departamento de Biologia Geral, Universidade Federal de Viçosa, Viçosa, MG, Brazil.

 

 

ABSTRACT

The soybean (Glycine max (L.) Merrill) frogeye leaf spot is caused by the fungus Cercospora sojina Hara and is a widespread disease in Brazil and other countries, causing severe losses in grain yield and also affecting seed quality. The availability of DNA markers linked to genes for resistance to this disease would accelerate breeding programs, particularly when other traits are also being evaluated. Bulked segregant analysis was applied to 3 F₂ populations derived from crosses between the resistant cultivars Parana, Cristalina and Uberaba, and the susceptible cultivar Bossier. In the cross 'Parana' x 'Bossier', 2 RAPD markers were identified, CSOPA1_800C and CSOPA2_1,250C, located at 4.4 ± 1.8 centiMorgans (cM) and 3.4 ± 1.7 cM respectively from the resistance locus. DNA fragments of similar molecular weight were observed in the population derived from the cross 'Cristalina' x 'Bossier' at 2.3 ± 1.2 and 4.7 ± 1.5 cM from the resistance locus, respectively. In the offspring of the cross 'Uberaba' x 'Bossier', a DNA fragment corresponding to marker CSOPA1_800C was detected at 5.6 ± 2.1 cM from the resistance locus. Although marker CSOPA2_1,250C was not observed in this population, an additional marker was detected (CSOUB1_1,100C) at 6.7 ± 2.2 cM from the resistance locus. The 1,250 bp fragment of CSOPA2_1,250C was cloned and converted into a SCAR marker, which amplified a single fragment whose size corresponded to the cloned segment of the crosses involving cultivars Cristalina and Parana. Markers CSOPA1_800C, CSOPA2_1,250C and CSOUB1_1,100C were mapped to soybean linkage group J with the aid of known SSRs linked to the Rcs3 locus, indicating that the RAPD and SCAR markers identified in our research also tag this resistance gene.

Key words: molecular markers, RAPD, SCAR, soybean, Cercospora sojina, frogeye leaf spot.

Received: July 19, 2000; accepted: June 6, 2002.

 

 

INTRODUCTION

Frogeye leaf spot, caused by the fungus Cercospora sojina Hara, is a worldwide important soybean disease, causing both yield losses and seed deterioration. The use of resistant cultivars is the most efficient and cost-effective means of controlling this disease, but the existence of several C. sojina races (Yorinori, 1989b) demands the permanent search for new sources of resistance and their incorporation into resistance breeding programs. Another factor to be considered is that the evaluation of soybean crops with regard to this disease is a time-consuming process, which requires expertise for the precise distinction between susceptible and resistant plants.

These problems can be better managed in breeding programs by identifying and using DNA markers linked to resistance genes. DNA markers are abundant and essentially independent from environmental conditions (Keim et al., 1989), and several research groups have been using this tool in breeding programs (Shoemaker et al., 1992; Young and Kelly, 1996; Young et al., 1998). In this paper, we report on the identification of DNA markers linked to soybean loci responsible for resistance to C. sojina in soybean cultivars Cristalina, Parana and Uberaba, and also on the confirmation of the resistance locus identity.

 

MATERIAL AND METHODS

Genetic material and crosses

Seeds of the soybean cultivars Bossier, Cristalina, Paraná and Uberaba were provided by the Soybean Breeding Laboratory (SBL) of the Department of Plant Sciences of the Federal University of Viçosa, Minas Gerais, Brazil. The Bossier cultivar is susceptible to the race 4 C. sojina fungus, while 'Cristalina', 'Parana' and 'Uberaba' are resistant to it. A number of allelism tests involving these resistant cultivars and/or their resistant progenitors indicate that they harbor the same resistance locus, Rcs3, which is also present in cultivar Davis (Arias et al., 1996).

Origin and cultivation of C. sojina isolate

The monosporic isolate (provided by the SBL) used in all inoculations was collected in the Alto Paranaíba region of the Brazilian State of Minas Gerais, and identified as race 4 (Machado et al., 1997), its isolation and cultivation being carried out as described by Veiga (1973) and Cordeiro (1986).

Evaluation of symptoms

The crosses performed were 'Cristalina' x 'Bossier' (CB), 'Parana' x 'Bossier' (PB), and 'Uberaba' x 'Bossier' (UB), with 'Bossier' as the male parent in all crosses. F₁ plants were selfed, and the F₂ seeds were planted in a greenhouse. In addition to the parents, 219 CB, 126 PB, and 124 UB F₂ plants were tested for resistance/susceptibility to C. sojina. The primary leaf from each plant was collected and kept at -80 °C. When the first trifoliolate leaf was fully expanded, the plants were inoculated with the pathogen by spraying approximately 4 x 10⁵ conidia on each plant, on the abaxial and adaxial sides of the leaves (Casela et al., 1979). The plants were then transferred and kept for 3 days in a mist chamber maintained at 20 to 22 °C and 95% relative humidity.

Symptoms were assessed 20 days after inoculation, based on the scale proposed by Yorinori (1989a). To suit our purpose better, the scale was modified as follows: grade 1 = leaf without lesions; 2 = 1 to 10% of leaf area infected (LAI); 3 = 11 to 25% LAI; 4 = 26 to 50% LAI; and 5 = more than 50% LAI. Plants with scores from 1 to 3 were considered resistant, and those which scored 4 and 5 were considered susceptible.

DNA bulks and amplification

In order to identify homozygous F₂ plants for the construction of DNA bulks, 20 F₃ seeds were taken from each of 30 F₂ resistant plants (with score 1) and planted in a greenhouse; the resulting F₃ plants were inoculated with the pathogen and evaluated for symptoms, as described above.

For each cross, two DNA bulks (Michelmore et al., 1991) were constructed, one containing DNA from 6 homozygous susceptible F₂ plants, and the other containing DNA from 6 resistant plants. The DNA was extracted by the method described by Doyle and Doyle (1990), and RAPD DNA amplification was carried out by the method of Williams et al. (1990), using primers from Operon Technologies (Alameda, CA, USA) and a model 9600 thermocycler (Perkin-Elmer, Norwalk, CT, USA). 1,200 primers were tested for DNA amplification. Amplification conditions were as follows: 40 cycles, each consisting of a 15 s denaturation step at 94 °C, a 30 s annealing step at 35 °C, and a 1 min extension step at 72 °C. After the 40^th cycle, a final extension step of 7 min at 72 °C was performed. The amplification products were separated on 1.2% agarose gel containing 0.2 mg/mL ethidium bromide, immersed in pH 8.0 TBE (90 mM Tris-borate buffer, 1 mM EDTA), and the DNA bands were observed under UV light and photographed; only the most intense and reproducible bands were used for analysis. Primers generating DNA bands which were polymorphic between the bulks were individually tested against the components of each bulk and then tested against the F₂ population, to determine the genetic distances between the markers and the resistance loci.

Development of SCAR markers

One of the RAPD bands linked to the resistance locus identified in population CB was excised from the gel, purified with the aid of the Glass Max^tm DNA Isolation Matrix System (BRL) and cloned in the vector pGEM-T Easy (Promega). White colonies were grown in 2 mL LB medium containing 100 mg/mL ampicillin, and the plasmid was purified with the QIA Prep Spin Miniprep kit (Qiagen). The clone was partially sequenced by automated sequencing using M13 universal primers. The sequence information was used to design two primers, each one containing 18 nucleotides including the sequence of the original RAPD primer.

The PCR reaction to amplify the SCAR marker consisted of 35 cycles, each one consisting of a 30 s step at 94 °C, a 1 min step at 62 °C, and a 1 min 30 s step at 72 °C. The amplified bands were analyzed as described for the RAPD amplification products.

Linkage analysis

Student's chi-square (c²) test was used to analyze the phenotypic segregation of the three populations and to determine possible linkages between the RAPD markers and the resistance loci.

For linkage analysis, 219 CB, 126 PB, and 124 UB F₂ plants were used, and the progenitors of these crosses were also tested with primer-pairs for SSR Satt-547 and Satt-431, which were shown to be linked to the Rcs3 locus in soybean linkage group J (Mian et al., 1999). Where polymorphic bands were detected, the contrasting bulks and the F₂ plants were also analyzed. The distances between the molecular marker loci and the resistance locus were estimated using version 3.0 of the MAPMAKER/EXP program (Lander et al., 1987; Lincoln et al., 1992), with a minimum lod score of 3.0 and a recombination setting of 50%.

 

RESULTS AND DISCUSSION

Identification of RAPD markers

Figure 1a and 1b show the two polymorphic DNA bands which distinguished the contrasting bulks from the PB cross. These bands were present in all resistant individuals of the bulks and absent in all susceptible individuals. These markers were designated CSOPA1_800C and CSOPA2_1,250C, because they are linked to a locus which controls resistance to C. sojina (CSO) originally detected in cultivar Parana (PA), contain approximately 800 and 1,250 bp, respectively, and are in the coupling-phase (C) with regard to the resistance gene. These two markers also co-segregated with resistance in the CB cross. Marker CSOPA1_800C also co-segregated with resistance in the UB cross, but locus CSOPA2_1,250C was monomorphic in that cross. A third marker, CSOUB1_1,100C, was detected only in the UB cross (Figure 1c).

 

 

The segregation ratio 3:1 (resistant:susceptible or band presence:absence) in the F₂ populations was consistent with the single-factor dominant inheritance of the resistance locus and of the RAPD markers in the CB and PB crosses (Table I). In the UB cross, resistance to C. sojina segregated at a 13:3 ratio (Table I), indicating that at least two independent loci control resistance in the Uberaba cultivar, one being dominant and the other recessive. Cordeiro (1986) also detected two resistance loci to C. sojina race 4 in a cross between 'Santa Rosa' and 'Bossier', 'Santa Rosa' being the progenitor of 'Uberaba'.

 

 

In view of the high c² values, the hypothesis of independent linkage (9:3:3:1) could be discarded, indicating that the RAPD markers were linked to the resistance locus (Table II). The estimates of the genetic distance between the RAPD locus CSOPA1_800C and the resistance locus varied between 2.3 cM for the CB cross and 5.6 cM for the UB cross. The estimated genetic distances between RAPD locus CSOPA2_1,250C and the resistance locus varied between 3.4 cM for the PB cross and 4.7 cM for the CB cross. CSOUB1_1,100C was 6.7 cM from the resistance locus.

 

 

It is conceivable that the locus tagged by CSOPA1_800C in all three crosses corresponds to locus Rcs3, a resistance locus previously identified in the cultivars Parana, Cristalina, Santa Rosa and Davis (Arias et al., 1996). To confirm this hypothesis, two SSR markers, Satt 431 and Satt 547, previously shown to be linked to locus Rcs3 (Mian et al., 1999), were tested in the three populations used in this study (Table I). The results show that, in the CB population, Satt 431 and Satt 547 co-segregated with resistance and with the CSOPA1_800C and CSOPA2_1,250C markers. However, in the PB population, both SSR markers were monomorphic, Satt 547 alone being polymorphic in the UB population and co-segregating with resistance and with the CSOPA1_800C and CSOUB1_1,100C markers. These SSR and RAPD marker data strongly suggest that the resistance locus we mapped in the three populations corresponds to the resistance locus Rcs3.

It is noteworthy that this conclusion could only be reached by the combined analysis of the two types of markers. In addition, our results confirm the usefulness of the integrated SSR map for soybean (Cregan et al., 1999) as a main guide for breeders and emphasize that the optimal use of molecular markers in plant breeding will depend on a specific analysis of each individual cross.

Conversion of RAPD marker into a SCAR

The DNA fragment corresponding to RAPD marker CSOPA2_1,250C was cloned and partially sequenced. Two 18-nucleotide-long primers were synthesized based on the sequencing data: SCARBG5F (5' GCC GTG AGA AAG GCG AAG 3') and SCARBG5R (5' AGC CGT GAA TTA TCC GAT 3'). These primers were tested in the CB and PB F₂ populations, and Figure 2 shows that the polymorphism of the amplifications is identical to the one revealed by the RAPD marker. This SCAR marker can be used for marker-assisted selection in programs aiming at the development of cultivars which are resistant to frogeye leaf spot, particularly when other traits are also being evaluated.

 

 

ACKNOWLEDGMENTS

The authors thank BID/FINEP and FAPEMIG for financial support. SMF was supported by a fellowship from CNPq.

 

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Send correspondence to E.G.de Barros. E-mail: ebarros@mail.ufv.br.