Identification of a RAPD marker linked to the Co-6 anthracnose resistant gene in common bean cultivar AB 136

The pathogenic variability of the fungus Colletotrichum lindemuthianum represents an obstacle for the creation of resistant common bean (Phaseolus vulgaris L.) varieties. Gene pyramiding is an alternative strategy for the development of varieties with durable resistance. RAPD markers have been proposed as a means to facilitate pyramiding of resistance genes without the need for multiple inoculations of the pathogens. The main aims of this work were to define the inheritance pattern of resistance present in common bean cultivar AB 136 in segregating populations derived from crosses with cultivar Rudá (susceptible to most C. lindemuthianum races) and to identify RAPD markers linked to anthracnose resistance. The two progenitors, populations F1 and F2, F2:3 families and backcrossderived plants were inoculated with race 89 of C. lindemuthianum under environmentally controlled greenhouse conditions. The results indicate that a single dominant gene, Co-6, controls common bean resistance to this race, giving a segregation ratio between resistant and susceptible plants of 3:1 in the F2, 1:0 in the backcrosses to AB 136 and 1:1 in the backcross to Rudá. The segregation ratio of F2:3 families derived from F2 resistant plants was 1:2 (homozygous to heterozygous resistant). Molecular marker analyses in the F2 population identified a DNA band of approximately 940 base pairs (OPAZ20940), linked in coupling phase at 7.1 cM of the Co-6 gene. This marker is being used in our backcross breeding program to develop Rudá-derived common bean cultivars resistant to anthracnose and adapted to central Brazil. Instituto de Biotecnologia Aplicada à Agropecuária (BIOAGRO), Universidade Federal de Viçosa (UFV), 36571-000 Viçosa, MG, Brasil. Send correspondence to A.L.A.M. E-mail: aalzate@alunos.ufv.br. EPAMIG, Departamentos de Biologia Geral, and Bioquímica e Biologia Molecular, UFV, Viçosa, MG, Brasil. 634 Alzate-Marin et al. (susceptible to most C. lindemuthianum races) and to identify RAPD markers linked to the resistance gene (Co-6) present in this cultivar. MATERIAL AND METHODS Source of C. lindemuthianum isolates and culture conditions Race 89 of C. lindemuthianum was collected in Minas Gerais State (Brazil) and corresponds to race alpha-Brazil (group alpha), part of a group of 25 races collected in different regions of Brazil and identified by Rava et al. (1994). The original inoculum was kindly provided by Dr. Carlos A. Rava and Dr. Aloisio Sartorato (CNPAF/EMBRAPA, Goiânia, GO, Brazil). To increase the amount of spores the isolates were cultivated for approximately 10 days in sterile medium containing common bean green pods. To confirm the identity of the isolates, they were inoculated on the 12 bean anthracnose differentials according to the method of Pastor-Corrales (1992).


INTRODUCTION
The common bean (Phaseolus vulgaris L.) is the main source of vegetable protein in most Latin American and African countries.The Brazilian population is the world's largest consumer, with an annual average intake of 22-23 kg/ person (Ventura and Costa, 1992).
Fungal diseases drastically affect common bean yield, and anthracnose, caused by the fungus Colletotrichum lindemuthianum (Sacc.& Magn.)Scrib., is among the main diseases of the common bean in Brazil and in other beangrowing regions of the world (Pastor-Corrales, 1985).This disease may cause yield losses of up to 100% when infected seeds are sown in areas with high relative humidity and mild temperatures (Peloso, 1992).
Although breeding the common bean for resistance to anthracnose by traditional methods has created several resistant cultivars (Singh et al., 1992;Vieira, 1983), new varieties have to be continually developed due to the high pathogenic variability of the fungus (Rava et al., 1994).Gene pyramiding is an alternative strategy for the development of varieties with durable resistance, but traditional breeding procedures are inefficient for such purpose due to the difficulties brought about by the need for multiple inoculations (Michelmore, 1995).Pyramiding of resistance genes assisted by molecular markers has been proposed as an alternative solution for this type of problem (Haley et al., 1993(Haley et al., , 1994;;Johnson and Gepts, 1994;Johnson et al., 1995;Young and Kelly, 1996a).
A number of common bean lines have been evaluated for resistance to many pathotypes of anthracnose at the Centro Internacional de Agricultura Tropical (CIAT, Cali, Colombia), and among them, cultivar AB 136, selected as one of the 12 international differential cultivars for anthracnose, is one of the most resistant (Pastor-Corrales, 1992).Cultivar AB 136 showed resistance to 25 pathotypes of C. lindemuthianum collected in several common bean-growing regions in Brazil (Rava et al., 1994).
Random amplified polymorphic DNA (RAPD) markers (Williams et al., 1990) have been successfully used in common bean breeding programs to tag resistance genes for several important diseases (Haley et al., 1993(Haley et al., , 1994;;Johnson and Gepts, 1994;Johnson et al., 1995;Young and Kelly, 1996a;Alzate-Marin et al., 1999a).This technique is being used in our common bean breeding program to facilitate the development of isolines containing disease resistance genes of interest.
The main aim of this work was to define the inheritance pattern of resistance present in cultivar AB 136 in segregating populations derived from crosses with cultivar Rudá Identification of a RAPD marker linked to the Co-6 anthracnose resistant gene in common bean cultivar AB 136 Ana Lilia Alzate-Marin 1 , Henrique Menarim 1 , José Mauro Chagas2 , Everaldo Gonçalves de Barros 1,3 and Maurilio Alves Moreira 1,4

Abstract
The pathogenic variability of the fungus Colletotrichum lindemuthianum represents an obstacle for the creation of resistant common bean (Phaseolus vulgaris L.) varieties.Gene pyramiding is an alternative strategy for the development of varieties with durable resistance.RAPD markers have been proposed as a means to facilitate pyramiding of resistance genes without the need for multiple inoculations of the pathogens.The main aims of this work were to define the inheritance pattern of resistance present in common bean cultivar AB 136 in segregating populations derived from crosses with cultivar Rudá (susceptible to most C. lindemuthianum races) and to identify RAPD markers linked to anthracnose resistance.The two progenitors, populations F 1 and F 2 , F 2:3 families and backcrossderived plants were inoculated with race 89 of C. lindemuthianum under environmentally controlled greenhouse conditions.The results indicate that a single dominant gene, Co-6, controls common bean resistance to this race, giving a segregation ratio between resistant and susceptible plants of 3:1 in the F 2 , 1:0 in the backcrosses to AB 136 and 1:1 in the backcross to Rudá.The segregation ratio of F 2:3 families derived from F 2 resistant plants was 1:2 (homozygous to heterozygous resistant).Molecular marker analyses in the F 2 population identified a DNA band of approximately 940 base pairs (OPAZ20 940 ), linked in coupling phase at 7.1 cM of the Co-6 gene.This marker is being used in our backcross breeding program to develop Rudá-derived common bean cultivars resistant to anthracnose and adapted to central Brazil.
(susceptible to most C. lindemuthianum races) and to identify RAPD markers linked to the resistance gene (Co-6) present in this cultivar.

Source of C. lindemuthianum isolates and culture conditions
Race 89 of C. lindemuthianum was collected in Minas Gerais State (Brazil) and corresponds to race alpha-Brazil (group alpha), part of a group of 25 races collected in different regions of Brazil and identified by Rava et al. (1994).The original inoculum was kindly provided by Dr. Carlos A. Rava and Dr. Aloisio Sartorato (CNPAF/EMBRAPA, Goiânia, GO, Brazil).To increase the amount of spores the isolates were cultivated for approximately 10 days in sterile medium containing common bean green pods.To confirm the identity of the isolates, they were inoculated on the 12 bean anthracnose differentials according to the method of Pastor-Corrales (1992).

Genetic material
Seeds from differential cultivar AB 136 and "carioca type" cultivar Rudá were provided by CNPAF/EMBRAPA (Goiânia, GO, Brazil) (Table I).Cultivar AB 136 was used as male progenitor and crossed in a greenhouse with cultivar Rudá.The populations were maintained in the greenhouse.

Genetic analyses and evaluation of disease symptoms
Spores of C. lindemuthianum race 89 were inoculated onto plants derived from the cross Rudá x AB 136.The following number of seeds were sowed in the greenhouse in a completely randomized design: 30 seeds from each progenitor, 30 F 1 seeds, 256 F 2 seeds and 60 seeds from each backcross (BC r and BC s ).Fourteen days after sowing the first expanded trifoliate leaf from each of the 466 plants was inoculated on the lower and upper leaf surfaces with spore suspensions (1.2 x 10 6 spores/ml), applied with a horse-hair paint brush.The plants were then incu-bated for seven days in a mist chamber, which was maintained at 20-22 o C and 100% relative humidity, after which the plants were evaluated using a 1-to 9-symptom scale (Rava et al., 1993).Resistant (R) phenotype was assigned to plants with no or limited symptoms (grades 1 to 3), whereas plants graded 4 or greater were considered to be susceptible (S).Before inoculation, primary leaves from all plants were collected and kept at -80 o C for DNA extraction.The phenotypic class frequencies obtained were tested for goodness-of-fit to theorical ratios with chisquare tests.
One hundred and eighty-eight F 2:3 families derived from F 2 resistant plants of cross Rudá x AB 136, consisting of 12 plants each, were evaluated for resistance/susceptibility to C. lindemuthianum race 89.Inoculation conditions and symptom evaluation were performed as before except that in this case spores were sprayed onto the plants with the aid of a De Vilbiss apparatus.

DNA bulks
DNA from homozygous F 2 resistant individuals was used to make two contrasting bulks (resistant/susceptible) according to Michelmore et al. (1991).Two hundred and ten primers (Operon Technologies, Alameda, CA, USA) were used to test the bulks.
Each amplification cycle consisted of one denaturation step at 94 o C for 15 s, one annealing step at 35 o C for 30 s and one extension step at 72 o C for 1 min.After 40 cycles an extra extension step was performed for 7 min at 72 o C. Amplification products were analyzed on 1.2% agarose gels immersed in TBE buffer (90 mM Tris-borate, 1 mM EDTA, pH 8.0) containing 10 mg/ml ethidium bromide.DNA bands were visualized under UV light and pho-

Linkage analyses
Chi-square analysis was used to test the phenotypic segregation of the F 1 , F 2 , F 2:3 , BC s and BC r populations.To determine the genetic distance between the RAPD marker and the resistance gene, 239 F 2 plants were used.The genetic distance between the marker and the resistance gene was determined with the aid of MAP-MAKER III (Lander et al., 1987) using a LOD score minimum of 3.0.

Segregation analyses
Results of inoculations confirmed that cultivar AB 136 is resistant and that cultivar Rudá is susceptible to C. lindemuthianum race 89 (Table II) under greenhouse conditions.Rava et al. (1994) demonstrated that cultivar AB 136 is resistant to 25 C. lindemuthianum races detected in Brazil.
The segregation ratio (resistant:susceptible) of 3:1 in the F 2 and the good fit to the expected (homozygote:heterozygote) ratio in the F 2:3 families derived from F 2 plants (Table II) indicate that resistance of AB 136 to race 89 is controlled by a single dominant gene.This gene was identified in cultivar Catrachita, a breeding line derived from AB 136 and named Co-6 (Young and Kelly, 1996b).Similar results were reported in previous work using the same resistance source (Vidigal, 1994;Alzate-Marin et al., 1999a).

Identification of RAPD markers
Co-6 has proven to be effective against a large number of Latin American isolates of C. lindemuthianum and it is an important resistance source used by breeding programs in Central America (Pastor-Corrales, 1985, 1992;Young and Kelly, 1996b;Rava et al., 1994).Young and Kelly (1997) identified two RAPD markers linked to, and flanking, the Co-6 locus in cultivar Catrachita: marker OPAH1 780 is linked in coupling-phase at 12.3 cM of the gene and marker OPAK20 890 is linked in repulsion phase at 7.1 cM of the gene.The band produced by OPAH1 780 is polymorphic between Rudá and AB 136, but in co-segregation analyses in the F 2 population it mapped at 23.7 cM of Co-6 (Table III, Figure 1).
We previously identified another marker (OPZ04 560 ) tightly linked in coupling to Co-6 (Alzate-Marin et al., 1999a).Using this marker it was possible to confirm that a single gene or a complex locus of tightly linked resistance genes present in cultivar AB 136 confers resistance to C. lindemuthianum pathotypes 73, 81, 89, and 64.The OPZ04 560 marker could be used in a marker-assisted breeding program to pyramid different resistance genes using cultivar AB 136 as a source for anthracnose resistance.In the case of the cross between cultivars Rudá and AB 136, however, OPZ04 560 was not polymorphic between the progenitors.For this reason it cannot be used in our breeding program aimed at the development of Rudá-derived bean varieties resistant to anthracnose.
One RAPD marker (OPAZ20 940 ) was identified in the F 2 plants inoculated with race 89.This marker, a 940-bp DNA band, is linked in coupling phase to the resistance gene   (Co-6) (Figure 2).Co-segregation analyses in the F 2 population revealed that marker OPAZ20 940 was located 7.1 cM from the resistance gene (Table III).The presence of this marker in cultivar Catrachita (Figure 3) strongly supports the idea that the gene studied in this work is the same (Co-6) as that reported by Young and Kelly (1996b).
To determine if the other 11 differential cultivars carry the molecular marker OPAZ20 linked to the Co-6 gene in cultivar AB 136, the DNA of these cultivars was extracted and amplified with the appropriate primers.Only differential cultivar AB 136 possessed the band OPAZ20 940 .The allelism studies conducted so far do not indicate the presence of alternative alleles for Co-6 in other differential cultivars for anthracnose (Young and Kelly, 1996b).
Marker OPAZ20 940 is being used to facilitate the development of Rudá-derived isolines containing the gene Co-6 (Alzate-Marin et al., 1999b), as well as to pyramid different resistance genes in common bean varieties as part of our breeding program toward the creation of commercial cultivars with long-lasting resistance to anthracnose.

ACKNOWLEDGMENTS
Research supported by grants from FAPEMIG (Brazilian Government).A. Alzate-Marin was the recipient of a post-doctoral fellowship from FAPEMIG and H. Menarim a fellowship from CNPq.

Figure 1 -Figure 3 -
Figure 1 -Electrophoretic analysis of amplification products obtained with primer OPAH1.The first lane corresponds to lambda DNA cut with EcoRI, BamHI and HindIII (size markers).Lanes 1 to 18 are as follows: 1, Rudá; 2, AB 136; 3-10, F 2 plants resistant to race 89; 11-18, F 2 plants susceptible to race 89.The arrow indicates a DNA band of 780 bp linked in coupling phase to the resistance gene.

Figure 2 -
Figure 2 -Electrophoretic analysis of amplification products obtained with primer OPAZ20.The first lane coresponds to lambda DNA cut with EcoRI, BamHI and HindIII (size markers).Lanes 1 to 18 are as follows: 1, Rudá; 2, AB 136; 3-10, F 2 plants resistant to race 89; 11-18, F 2 plants susceptible to race 89.The arrow indicates a DNA band of 940 bp linked in coupling phase to the resistance gene.

Table I -
Phenotypic characteristics of common bean progenitors Rudá and AB 136.

Table III -
Linkage analysis between molecular markers and resistance genes in crosses involving cultivar AB 136.
a Distance in centimorgans in relation to Co-6.b Resistance gene present in cultivar AB 136.

Table II -
Segregation for resistance to race 89 of Colletotrichum lindemuthianum in the cross Rudá x AB 136.