Euphytica (2006) 151:411–419 DOI 10.1007/s10681-006-9164-x
Inheritance of anthracnose resistance in the common bean cultivar Widusa M. Celeste Gon¸calves-Vidigal · James D. Kelly
Received: 20 October 2004 / Accepted: 5 April 2006 C Springer Science + Business Media B.V. 2006
Abstract Snap bean (Phaseolus vulgaris L.) cultivar, Widusa, was crossed to Michigan Dark Red Kidney (MDRK), Michelite, BAT 93, Mexico 222, Cornell 49–242, and TO cultivars to study the inheritance of resistance to anthracnose in Widusa. The segregation patterns observed in six F2 populations supported an expected 3R:1S ratio suggesting that Widusa carries a single dominant gene conditioning resistance to races 7, 65, 73, and 453 of Colletotrichum lindemuthianum, the causal organism of bean anthracnose. Allelism tests conducted with F2 populations derived from crosses between Widusa and Cornell 49–242 (Co-2), Mexico 222 (Co-3), TO (Co-4), TU (Co-5), AB 136 (Co-6), BAT 93 (Co-9), and Ouro Negro (Co-10), inoculated with races 7, 9, 65 and 73, showed a segregation ratio of 15R:1S. These results suggest that the anthracnose resistance gene in Widusa is independent from the Co-2, Co-3, Co-4,Co-5, Co-6, Co-9, and Co-10 genes. A lack of segregation was observed among 200 F2 individuals from the cross Widusa/MDRK, and among 138 F2 individuals from the cross Widusa/Kaboon inoculated with race 65, suggesting that Widusa carries an allele at the M. C. Gon¸calves-Vidigal Departamento de Agronomia, Universidade Estadual de Maring´a, Av. Colombo, 5790, 87020-900, Maring´a, PR, Brazil J. D. Kelly () Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI, 48824, USA e-mail:
[email protected]
Co-1 locus. We propose that the anthracnose resistance allele in Widusa be named Co-15 as Widusa exhibits a unique reaction to race 89 compared to other alleles at the Co-1 locus. RAPD marker A181500 co-segregated in repulsion-phase linkage with the Co-15 gene at a distance of 1.2 cM and will provide bean breeders with a ready tool to enhance the use of the Co-15 gene in future bean cultivars. Keywords Allelic series . Colletotrichum lindemuthianum . Molecular marker . Phaseolus vulgaris Abbreviations MDRK Michigan dark red kidney RAPD Random amplified polymorphic DNA
Introduction Anthracnose, caused by Colletotrichum lindemuthianum (Sacc. & Magn.) Scrib., is one of the most widespread and economically important fungal diseases of common bean (Phaseolus vulgaris L.). Genetic resistance is the most effective method of controlling anthracnose in common bean and as new resistance sources become available (Mahuku et al., 2002) their genetic characterization is essential to ensure novelty from previously characterized sources. Ten independent resistance loci, Co-1 to Co-10, that condition resistance to anthracnose have been identified and mapped Springer
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to different linkage groups of the bean genome and allelic series are known to exist at the Co-1, Co-3 and Co-4 loci (Kelly & Vallejo, 2004). The 12-member differential series (Pastor-Corrales, 1991) used to differentiate races of C. lindemuthianum has proved to be a valuable source of resistance genes for breeders. The differential series was established with limited knowledge of the genes present in individual cultivars. The majority of the resistance genes present in the differential series have been characterized with the exception of Widusa, a European snap bean (Drijfhout & Davis, 1989). Widusa was previously considered to belong to the Andean gene pool of P. vulgaris, but recent pedigree evidence suggests that its origins are Middle American (Myers, personal communication). Widusa, with the binary code number 16 (Pastor-Corrales, 1991), is the differential cultivar that separates the widespread race 73 (Balardin et al., 1997) from race 89 (alpha-Brazil) found in Brazil (Balardin et al., 1990; Thomazella et al., 2000), and Canada (Tu, 1994). As the name suggests, race alpha-Brazil originated in Brazil, was first characterized by Fouilloux (1979) in France, and renamed race 89 using the standard differential series (Melotto et al., 2000). Despite the breakdown of resistance to race 89, Widusa is resistant to 30 races of C. lindemuthianum in Brazil (Alzate-Marin et al., 2002). Previous studies on the inheritance of anthracnose resistance in Widusa confirmed the presence of a single dominant gene (Alzate-Marin et al., 2002; Gon¸calvesVidigal et al., 2003), but Ferreira et al. (2003) reported the presence of two independent genes conferring resistance to race 38. Given the importance of the resistance in Widusa to bean breeders in Brazil, this study was undertaken to determine the inheritance of resistance to anthracnose in Widusa; to verify independence of the resistance from other characterized genes in allelism studies; and to develop markers linked to the resistance in Widusa.
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BAT 93 (Co-9) and Ouro Negro (Co-10), obtained from the germplasm bank of NUPAGRI (N´ucleo de Pesquisa Aplicado a` Agricultura), at the Universidade Estadual de Maring´a Brazil, were used as pollen parents in crosses with Widusa to develop segregating populations to characterize the genetic resistance in Widusa (Table 1). Five races, 7, 9, 65, 73, and 453 of C. lindemuthianum, incompatible with Widusa, were selected to provide differential reaction in the segregating populations generated. The reaction of all five races on the parental materials and the differential cultivars is shown in Table 1. The inheritance of anthracnose resistance in Widusa was verified in six segregating populations where compatible races were chosen to produce a S × R reaction with the following six cultivars: MDRK (susceptible to race 7), Cornell 49–242 (susceptible to race 73), TO (susceptible to race 453), Michelite, Mexico 222, and BAT 93 (susceptible to race 65; Table 2). Tests for allelism were conducted for eight loci: Co-1, Co-2, Co-3, Co-4, Co-5, Co-6, Co-9 and Co-10 in 15 segregating populations (Table 3). The populations were derived from the crosses between Widusa and the differential cultivars in which the anthracnose race was chosen to provide an incompatible R × R reaction with both parents. Certain populations were inoculated with additional races of the pathogen to confirm reaction of segregating progeny (Tables 2 and 3). Progeny of four F2 populations of Widusa with MDRK, Cornell 49–242, TO, and BAT 93 were harvested to generate F2:3 families to confirm segregation ratios in the F2 generation. The F2:3 families of the Widusa/Cornell 49–242 cross was also used as a mapping population for co-segregation studies with RAPD markers putatively linked to the resistance in Widusa. Ten plants from each F2:3 family were inoculated to confirm the F2 genotype, and based on the number of susceptible plants observed in individual families, F2 individuals were classified as either homozygous dominant, heterozygous, or homozygous recessive.
Materials and methods Preparation of C. lindemuthianum Plant material Seven members of the anthracnose differential set of 12 cultivars each possessing different anthracnose (Co-) resistance genes, Michelite, MDRK (Co-1), Cornell 49-242 (Co-2), Mexico 222 (Co-3), TO (Co-4), TU (Co-5), PI 207262 (Co-43 + Co-9), and two genotypes Springer
Races 7, 65, 73, and 453 of C. lindemuthianum from the collection of the NUPAGRI, Agronomy Department (Universidade Estadual de Maring´a; NUPAGRI code for isolates used: CL 7–10, CL 65-9, CL 73-3, and CL 453-2, respectively) were collected in the Brazilian state of Paran´a and characterized by
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Table 1 Genotypes, gene pool origin, anthracnose resistance genes, binary code number and disease reaction of cultivars to different races of Colletotrichum lindemuthianum Races of C. lindemuthianum Cultivar
Gene pool
Resistance gene
Michelite Widusa MDRK Perry Marrow Kaboon AND 277 Cornell 49–242 Mexico 222 TO TU PI 207262 AB 136 BAT 93 Ouro Negro
MAa A A A A A MA MA MA MA MA MA MA MA
Unknown Unknown Co-1 Co-13 Co-12 Co-14 Co-2 Co-3 Co-4 Co-5 Co-43 , Co-9 Co-6 Co-9 Co-10
Binary code no.b 1 16 2 4 32 – 8 64 256 512 128 1024 – –
7
9
65
73
453
S Rc S S R S R R R R R R R R
S R R R R R S R R R R R R R
S R R R R R R S R R R R S S
S R R R R R S S R R R R R R
S R R S R R R S S R S R S R
MA = Middle American, A = Andean Melotto et al., (2000) c R = Resistant; S = Susceptible; MDRK = Michigan Dark Red Kidney a
b
Table 2 Reaction of F2 and F2:3 populations, observed and expected ratios of resistant (R-) and susceptible (rr) plants inoculated with different races of C. lindemuthianum in R × S crosses with the resistant parent, Widusa Phenotypic evaluation in the F2
Phenotypic evaluation in F2:3 families
Crosses with Widusaa
Race
Expected ratio
R-
rr
χ2
P value
RR
R-
rr
χ2
P value
MDRK Cornell 49–242 TO BAT 93 Michelite M´exico 222
7 73 453 65 65 65
3:1 3:1 3:1 3:1 3:1 3:1
164 119 151 252 70 85
57 40 43 84 24 29
0.074 0.002 0.435 0.0 0.014 0.012
0.79 0.96 0.51 1.0 0.91 0.91
23 25 50 26 – –
43 43 101 36 – –
24 23 43 20 – –
0.905 0.834 0.659 0.350 – –
0.90 0.83 0.65 0.80 – –
a
Widusa used as female parent; MDRK = Michigan Dark Red Kidney
(Thomazella et al., 2000), and race 9 (Balardin et al., 1997) was obtained from Michigan State University (MSU isolate code: CL Hond. 9.1). Cultures from each of the races were incubated on Petri dishes containing either Mathur’s (Mathur et al., 1950), PDA (potatodextrose agar) or bean pod agar (Tuite, 1969). These cultures were incubated at 25 ◦ C for 14 days and were inoculated on the anthracnose differential series to confirm their race classification. Inoculation of the parents, F1 , F2 , and F2:3 families of each cross were carried out separately to prevent contamination. Fifteen parental and F1 individuals from each cross were inoculated and the number of F2 individuals inoculated varied by cross
and is shown in Tables 2 and 3. The protocol for inoculation was as follows: 14-day-old bean plants with fully developed first trifoliate leaves were spray-inoculated with a spore suspension (1.2 × 106 spores per ml) of each race of C. lindemuthianum. After inoculation, the plants were maintained in a mist chamber for 48 h at 20 ◦ C ± 2 ◦ C, under controlled lighting (12 h daylight) and approximately 100% relative humidity. Seedlings were evaluated for their disease reaction using a scale of 1 to 9 (Balardin et al., 1990) 7d after inoculation. Plants with disease reaction scores of 1–3 were considered resistant, whereas plants that were rated 4–9 were considered susceptible. Springer
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Euphytica (2006) 151:411–419 Table 3 Allelism tests for genetic characterization of anthracnose resistance in Widusa. Reaction of 16 F2 populations, observed and expected ratios of
resistant (R) and susceptible (S) plants to inoculation with different races of C. lindemuthianum in R × R crosses with Widusa Observed ratio
Expected ratio
Crosses with Widusaa
Race
Resistance gene
R
S
R:S
χ2
P value
Cornell 49–242 TO TU BAT 93 AB 136 Mexico 222 MDRK Kaboon AB 136 PI 207262 BAT 93 PI 207262 TO TU AB 136 Ouro Negro
7 7 7 7 7 9 65 65 65 65 73 73 73 73 73 73
Co-2 Co-4 Co-5 Co-9 Co-6 Co-3 Co-1 Co-12 Co-6 Co-43 , Co-9 Co-9 Co-43 , Co-9 Co-4 Co-5 Co-6 Co-10
137 174 216 229 65 139 200 138 82 269 120 352 200 92 121 119
11 12 13 17 4 10 0 0 5 18 9 6 11 5 8 8
15:1 15:1 15:1 15:1 15:1 15:1 – – 15:1 15:1 15:1 63:1 15:1 15:1 15:1 15:1
0.353 0.013 0.124 0.183 0.024 0.054 – – 0.037 0.001 0.116 0.029 0.387 0.199 0.005 0.005
0.55 0.92 0.72 0.67 0.88 0.82 – – 0.85 0.98 0.73 0.86 0.53 0.66 0.98 0.98
a
Widusa used as female parent; MDRK = Michigan Dark Red Kidney
RAPD Analysis
Linkage analysis
Linkage between RAPD marker(s) and the resistance gene(s) in Widusa was tested in 91 F2:3 families from the cross Widusa/Cornell 49–242. Two contrasting bulks were formed using bulked segregant analysis procedure (Michelmore et al., 1991) with DNA from 6 homozygous resistant F2 individuals, and 6 homozygous susceptible F2 plants derived from the mapping population (Widusa/Cornell 49–242) after inoculating F2:3 families with race 73. Prior to inoculation with C. lindemuthianum, tissue was collected for DNA extraction from young primary leaves, approximately 6 days post-emergence, from greenhouse-grown plants. The DNA extraction method followed the procedure of Afanador et al. (1993). Amplification reactions were performed similar to that described by Young and Kelly (1996) using random primers (Operon Technologies, Alameda, CA). The amplification was carried out in a thermal cycler (MJ Research Inc., Waltham, MA) using the profile: 3 cycles 94 ◦ C for 1 min, 35 ◦ C for 1 min, 72 ◦ C for 2 min; 34 cycles 94 ◦ C for 10 s, 40 ◦ C for 20 s, 72 ◦ C for 2 min; 1 cycle 72 ◦ C for 5 min. Those primers showing correspondence with resistance in the parents and the R and S bulks were used to screen the entire mapping population.
The phenotypic segregation was analyzed in the F2 population, and F2:3 families derived from the Widusa/Cornell 49–242 cross using the Chi-square test. Linkage analyses were performed using the computer software Mapmaker (Lander et al., 1987). In addition, the Linkage-1 computer program was used to determine linkage distance in centimorgans (cM) between loci using the Kosambi’s function.
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Results Data on the gene pool origin of Widusa and other differential cultivars and their disease reaction to the races 7, 9, 65, 73, and 453 are shown in Table 1. Widusa, Kaboon (Co-12 ), TU (Co-5) and AB 136 (Co-6) were the only genotypes resistant to these five races of C. lindemuthianum tested. Results of the inheritance studies with Widusa are shown in Table 2. The data support an expected 3R:1S ratio in all six F2 populations from the R × S crosses of Widusa with MDRK (p = 0.79), Cornell 49–242 (p = 0.96), TO (p = 0.51), BAT 93 (p = 1.0), Mexico 222 (p = 0.91), and Michelite (p = 0.91). The results indicated that Widusa carries a
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single dominant gene that confers resistance to races 7, 65, 73, 453 of C. lindemuthianum. The segregation ratios of the F2 populations were confirmed in F2:3 families, which showed a good fit to a 1: 2:1 segregation ratio in crosses of Widusa with MDRK (p = 0.90), Cornell 49–242 (p = 0.83), TO (p = 0.65), and BAT 93 ( p = 0.80). Results of allelism tests for independence of the single dominant resistant gene in Widusa are shown in Table 3. Segregation ratios that fit a 15R:1S (R-:rr) ratio were observed in 13 of the 16 F2 populations evaluated. These data would suggest that the single dominant gene in Widusa is independent from the Co-2, Co-3, Co-4, Co-5, Co-6, Co-9, and Co-10 genes in Cornell 49242, Mexico 222, TO, TU, AB 136, BAT 93 and Ouro Negro, respectively (Table 3). A 63R:1S ratio observed in the cross of Widusa with PI 207262 inoculated with race 73, suggested that three genes were segregating, which supported the independence of resistance in Widusa from the Co-43 and Co-9 resistance genes present in PI 207262. No segregation was observed among 200 individuals in the F2 population of Widusa and MDRK (Co-1), inoculated with race 65. These data would suggest that Widusa carries another allele at the Co-1 locus as the resistance factor in Widusa segregated as a single dominant gene in the R × S cross with MDRK inoculated with race 7 (Table 3). A similar lack of segregation was observed among 138 F2 individuals in the cross of Widusa with Kaboon (Co-12 ), adds further support for the presence in Widusa of another allele at the Co-1 locus (Table 3). Widusa appears to carry a new allele at the Co-1 locus, since it has a different resistance spectrum from all other characterized Co-1 alleles (Balardin et al., 1997; Table 1) based on its position in the differential series (Melotto et al., 2000). We propose that the anthracnose resistance allele conditioning resistance to races 7, 9, 65, 73, and 453 in Widusa be designated Co-15 . Identification of RAPD markers The RAPD marker designated OA181500 , generated by the 5 -AGGTGACCGT-3 decamer, was found to be tightly linked at distance of 1.2 cM from the resistance gene in Widusa (Fig. 1). This marker co-segregated in repulsion phase linkage with the resistant phenotype in 91 F2 individuals derived from the cross Widusa/Cornell 49–242, inoculated with race 73. Only one recombinant individual was observed.
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Discussion Widusa is one of the last anthracnose differential cultivars to be characterized. In six segregating populations, Widusa was shown to carry a single dominant gene conditioning resistance to races 7, 65, 73 and 453 of C. lindemuthianum (Table 2). Similar results were observed in the cross between Widusa (R) and Ruda (S) inoculated with race 65 (Alzate-Marin et al., 2002). However, data from the R × S cross of Widusa/Xana (341 F2 individuals) inoculated with race 38 suggests the presence of two independent resistance genes, one dominant and the other recessive in Widusa (13:3, p = 0.99; Ferreira et al., 2003). Contradictory results on the number of genes controlling anthracnose resistance are not uncommon in the published literature as the number of effective resistance genes that segregate after inoculation with different races of C. lindemuthianum can vary. Two gene and three gene models for the resistance in G 2333 have been reported in the literature (Kelly & Vallejo, 2004) since different races defeat different resistance genes in the host. For example, after inoculation with race 65, we showed that PI 207262 had a single gene for resistance similar to data reported by Mendez-Vigo et al. (2005) based on inoculation with race 38. However, previous workers reported the presence of two genes (Co-43 , Co-9) in PI 207262 (Kelly & Vallejo, 2004), similar to our findings after inoculation with race 73. Race 65 defeats the Co-9 gene, race 38 defeats the Co-43 allele, whereas race 73 is not pathogenic on either gene in PI 207262. We have clearly demonstrated that a single dominant gene is segregating in Widusa after inoculation with anthracnose races 7, 65, 73 and 453 which raises the question which of the two genes reported by Ferreira et al. (2003) might correspond with the gene identified in the current study. In the absence of direct inoculation of the same segregating populations with race 38, the answer will be speculative. The recessive gene detected by Ferreira et al. (2003) in Widusa after inoculation with race 38 may be the most likely candidate for the following reasons. Resistance to anthracnose in common bean is conditioned in most instances by independent dominant genes but recessive resistance has also been reported (Kelly & Vallejo, 2004). Recessive resistance has been observed at the multiallelic Co-1 locus after inoculation with virulent Andean races such as 130 (beta). Recessive resistance appears as a reversal of dominance resulting from dominance interaction among members of the Springer
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Fig. 1 Electrophoretic analysis of amplification products obtained with OA181500 RAPD marker. Lanes are as follows: 1, molecular weight marker (100 bp ladder); 2, Widusa (resistant); 3, Cornell 49–242 (susceptible); 4, resistant bulk; 5, susceptible
bulk; 6–11, F2 plants resistant to race 73; 12–17, F2 plants susceptible to race 73. The arrow indicates a DNA band of 1500 bp linked in repulsion phase to the resistance gene in Widusa
allelomorphic Co-1 series (Muhalet et al., 1981). The recessive resistance reported by Ferreira et al. (2003) in Widusa after inoculation with the Andean race 38 may be one such example. The Co-1 allele in MDRK appears to rank high in the order of dominance among alleles at the Co-1 locus, but given it susceptibility to Andean races 38 and 130, resistance appears to be recessive in crosses with other resistant alleles at the Co-1 locus that are weaker (less dominant) than the susceptible Co-1 allele (Melotto & Kelly, 2000). Published data provide evidence for a recessive resistance allele, that after inoculation with other races, segregates in a dominant (3R:1S) fashion (Muhalet et al., 1981). In the current study, no susceptible individuals were detected among 200 F2 individuals from the cross of neither Widusa/MDRK nor among 138 F2 individuals from the cross of Widusa/Kaboon, inoculated with race 65 (Table 3). This evidence supports the presence, in Widusa, of another allele at the Co-1 locus. Widusa appears to possess a unique allele for resistance different from the alleles present in other characterized Andean genotypes (Table 1). MDRK (Co-1), Perry Marrow (Co-13 ), Kaboon (Co-12 ) and AND277 (Co-14 ; Alzate Marin et al., 2003a) are resistant to race 89, whereas Widusa is susceptible which would provide supporting evidence that Widusa has a different allele at the Co-1 locus. The Co-15 allele present in Widusa is most probably the recessive resistance gene in the cross Widusa/Xana detected with race 38. Race 38 defeats all Co-1 alleles in the differential series: Co-1 (MDRK, no. 2), Co-13 (Perry Marrow; no. 4) and Co-12 (Kaboon,
no. 32), except the Co-15 gene in Widusa (no.16). If the Co-15 ranks lower in the order of dominance among the Co-1 alleles, then one would expect to see a recessive resistant reaction after inoculation with race 38 in crosses with the susceptible Co-1 allele. Based on the literature, there appears to be two independent anthracnose resistance genes in Widusa one dominant and the other recessive. We suggest that the recessive gene (Ferreira et al., 2003) is the same single dominant gene that was detected after inoculation with races 7, 65, 73, and 453 in the current study. The Co-15 allele present in Widusa is most probably the same dominant gene described by Alzate-Marin et al. (2002) as that gene was also independent of the Co-4 locus as we report below (Table 3). Data from allelism tests suggest independence between the Co-15 allele present in Widusa and the Co-2, Co-4, Co-5, Co-6, and Co-9 genes in the differential cultivars. Two gene segregation ratios (15R:1S) were observed in the five R × R crosses between Widusa with Cornell 49-242 (Co-2), TO (Co-4), TU (Co-5), AB 136 (Co-6) and BAT 93 (Co-9), based on inoculation with race 7 (Table 3). Further confirmation of an independent dominant gene in Widusa comes from crosses with TO, TU and BAT 93 inoculated with race 73 where two-gene independent ratios were observed. Additional support for independence of the Co-15 gene in Widusa from the Co-4 locus was also observed in R × R crosses with TO and SEL 1308 (Co-42 ; Alzate-Marin et al., 2002). A 3-gene segregation ratio (63R:1S, p = 0.72) was observed in the F2 population of the cross
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Widusa/PI 207262 inoculated with race 73. The segregation ratio supports the presence of three independent dominant genes with one gene (Co-15 ) in Widusa, and two genes Co-43 and Co-9 in PI 207262 (AlzateMarin et al., 2002; Kelly & Vallejo, 2004; Table 3). The two genes (15R:1S) ratio observed in the Widusa/PI 207262 cross after inoculation with race 65 is similarly explained as race 65 overcomes the Co-9 in PI 207262 (Table 3), thus no allelism was observed in this cross. The single gene (3R:1S) ratio of Widusa/BAT 93 confirmed that Co-9 is defeated by race 65 (Table 2). Race 65 used by Alzate-Marin et al. (2003c), however, generated a different reaction to the one observed in this study, since race 65 used by Alzate-Marin et al. (2003c) did not overcome the Co-9 gene in BAT 93 and allelism was observed with the Co-9 gene in crosses between Widusa with PI 207262 and BAT 93. In Brazil, genetic variability was verified among different isolates of race 65 analyzed with RAPD markers, demonstrating an intra-race molecular variability in this race (Thomazella, 2004). Consequently the isolates of race 65 used in this study were different from those utilized by Alzate-Marin et al. (2002, 2003c) in Minas Gerais, Brazil. Therefore, the putative allelism between a second gene in Widusa and the Co-9 gene (AlzateMarin et al., 2001, 2003c) cannot be confirmed. Other researchers have observed variability between isolates of race 65. Rodr´ıgues-Su´arez et al. (2005) observed that race 65b (same race used in current study) from Brazil revealed greater virulence on breeding lines A321 and A493 than race 65 from the collection maintained at the Michigan State University (Balardin et al., 1997). Since the four previously described races (7, 65, 73, 453) were compatible on the differential cultivar Mexico 222 (Co-3), race 9 was chosen to conduct the allelism test with Widusa as race 9 is incompatible on the Co-3 gene. The two gene (15R:1S) ratio observed in the Widusa/Mexico 222 cross after inoculation with race 9 indicates independence between the Co-3 and the Co-15 allele in Widusa (Table 3). The lack of allelism between Widusa and the Co-3 would also imply an absence of allelism with Co-9, since previous work showed that Co-3 and Co-9 were allelic (Mendez-Vigo et al., 2005). Inoculations of F2 populations of Widusa with race 38 also confirm independence between genes in Widusa and the Co-2, Co-3, Co-5 and Co-9 genes (Ferreira et al., 2003). The independence with the Co-9 gene is contradicted by the findings of Alzate-Marin et al. (2001) who showed allelism be-
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tween Widusa and PI 207262 in an F2 population of 170 individuals inoculated with race 65. Since there are two genes in PI 207262 (Co-43 , Co-9), they confirmed that the Co-9 gene was complementary with Widusa in crosses with BAT 93 (Alzate-Marin et al., 2003c) and not with the Co-4 alleles in TO and SEL 1308 (AlzateMarin et al., 2002). Since race 65 used by Alzate-Marin et al. (2001, 2002, 2003c), did not overcome the Co-9 gene in BAT 93 and allelism was observed with the Co-9 gene in crosses between Widusa with PI 207262 and BAT 93, the putative allelism between the second gene in Widusa and the Co-9 gene cannot be confirmed based on the reaction to race 65 used in the current study. Additional allelism tests conducted with F2 populations derived from crosses Widusa/AB 136 and Widusa/Ouro Negro, demonstrated that segregation patterns fit a 15R:1S ratio, indicating that the dominant gene, present in Widusa, is independent of the genes Co-6, in AB 136 and Co-10 in Ouro Negro. Similar results were obtained from the cross Widusa/Ouro Negro (Alzate-Marin et al., 2003b). In both studies the F2 populations were inoculated with race 73. A similar lack of allelism was observed in the cross Widusa with Michelite, inoculated with race 64. The segregation fitted a 15:1 ratio (p = 0.45), indicating the presence of two independent dominant genes (Gon¸calves-Vidigal et al., 2005). Complementation between gene(s) in Widusa and other known Co genes have been described (Ferreira et al., 2003; Alzate-Marin et al., 2001) but cannot be confirmed in the present study as we only detected a single dominant gene in Widusa after inoculation with races 7, 9, 65, 73, and 453. The complementation in Widusa could exist between the other gene(s) detected in those studies after inoculation with different races. According to Ferreira et al. (2003), Widusa has putative alleles at the resistance loci in Michelite, Co-4 (TO) and Co-6 (AB 136) based on a lack of segregation in the F2 populations, derived from crosses of Widusa with TO, Michelite, and AB 136. We were unable to support or confirm such allelism as other genes are being evaluated in inoculations with different races of C. lindemuthianum used in these studies. The combined results of the allelism tests (Table 3) support the hypothesis that only a single gene confers resistance to anthracnose in Widusa, and that gene is independent of the other reported genes Co-2, Co-3, Co-4, Co-43 , Co-5, Co-6, Co-9, and Co-10 (Kelly & Vallejo, 2004). A lack of segregation in crosses with Springer
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MDRK (Co-1) and Kaboon (Co-12 ) when inoculated with race 65, suggests that Widusa possesses an allele at the Co-1 locus. Different resistance alleles at the Co-1 locus have been characterized in the following cultivars belonging to the Andean gene pool: Kaboon (Co12 ), Perry Marrow (Co-13 ), AND 277 (Co-14 ), Widusa (Co-15 ), and Jalo EEP 558 (Co-1). Jalo carries the same allele at the Co-1 locus as MDRK, and A193 previously considered as having independent genes (Kelly & Vallejo, 2004; Melotto & Kelly, 2000; Mendoza et al., 2001; Alzate-Marin et al., 2003a; Gon¸calves-Vidigal et al., 2003; Vallejo et al., 2003). Since Widusa has a different resistance spectrum from all other characterized Co-1 alleles (Table 1), these data would indicate that Widusa carries a new allele at this locus. Therefore, we propose that the anthracnose resistance allele in Widusa conditioning resistance to races 7, 9, 65, 73 and 453 be designated as Co-15 . Identification of RAPD markers The RAPD marker OA181500 was identified using bulked segregant analysis of F2 individuals from the cross between Widusa and Cornell 49-242, inoculated with race 73 (Fig. 1). The marker was found closely linked in repulsion phase to the Co-15 resistant gene in Widusa. Other workers Young and Kelly (1997) found a RAPD marker in repulsion phase with Co-1 locus and Mendoza et al. (2001) reported an AFLP marker tightly linked at 2.7cM from Co-1, in repulsion phase. According to Haley et al. (1994), selection based on repulsion–phase markers (linked with the allele conferring susceptibility), yields a greater proportion of homozygous resistant plants, than the selection based on a coupling-phase (linked with the allele conferring resistance), even at greater recombination frequencies between marker, and resistance loci. The OA181500 marker, linked at distance of 1.2 cM, should be useful in marker-assisted selection for the introgression of Co-15 into susceptible germplasm and could improve the effectiveness of resistance gene pyramiding for anthracnose in bean breeding programs. Acknowledgements M.C. Gon¸calves-Vidigal recognizes financial support from CAPES, Brazil. The authors thank Halima Awale and Veronica Vallejo for their assistance in this research, and Maeli Melotto for critical review of the manuscript.
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Euphytica (2006) 151:411–419
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419 Muhalet CS, Adams MW, Saettler AW, Ghaderi A (1981) Genetic system for the reaction of field beans to beta, gamma, and delta races of Colletotrichum lindemuthianum. J Amer Soc Hort Sci 106:601–604 Pastor-Corrales MA (1991) Estandarizaci´on de variedades diferenciales y de designaci´on de razas de Colletotrichum lindemuthianum. Phytopathology 81:694 Rodr´ıguez-Su´arez C, Pa˜neda A, Campa A, Ferreira JJ, Gir´aldez R (2005) Anthracnose resistance spectra of breeding lines derived from the dry bean landrace Andecha. Annu Rept Bean Improv Coop 48:72–73 Thomazella C (2004) Identifica¸ca˜ o de ra¸cas e diversidade gen´etica de Colletotrichum lindemuthianum (Sacc. et Magn.) Scrib. em feijoeiro (Phaseolus vulgaris L). PhD Thesis, Universidade Estadual de Maring´a, Maring´a, Paran´a, Brazil Thomazella C, Gon¸calves-Vidigal MC, Vida JB, Vidigal Filho PS, Rimoldi F (2000) Identification of Colletotrichum lindemuthianum races in Phaseolus vulgaris L. Annu Rept Bean Improv Coop 43:82–83 Tu JC (1994) Occurrence and characterization of the alpha-Brazil race of bean anthracnose (Colletotrichum lindemuthianum) in Ontario. Can J Plant Pathol 16:129–131 Tuite J (1969) Plant Pathological Methods. Fungi and Bacteria. Burgess Pub. Co., Minneapolis, MN Vallejo VA, Awale HE, Kelly JD (2003) Characterization of the anthracnose resistance in the Andean bean cultivar Jalo EEP 558. Annu Rept Bean Improv Coop 46:179–180 Young RA, Kelly JD (1996) RAPD markers flanking the Are gene for anthracnose resistance in common bean. J Amer Soc Hort Sci 121:37–41 Young RA, Kelly JD (1997) RAPD markers linked to three major anthracnose resistance genes in common bean. Crop Sci 37:940–946
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